The basic helix-loop-helix-zipper transcription factor USF1 regulates expression

The Basic Helix-Loop-Helix-Zipper Transcription Factor USF1Regulates Expression of the Surfactant Protein-A Gene*

(Received for publication,April 17,1997,and in revised form,June 25,1997)

Erwei Gao?,Ying Wang?,Joseph L.Alcorn?,and Carole R.Mendelson?§?

From the Departments of §Biochemistry and ?Obstetrics-Gynecology,The University of Texas Southwestern Medical Center at Dallas,Dallas,Texas 75235-9038

Expression of the rabbit pulmonary surfactant pro-tein A (SP-A)gene is lung-specific,occurs primarily in type II cells,and is developmentally regulated.We pre-viously identified two E-box-like enhancers,termed the distal binding element (DBE)and proximal binding ele-ment (PBE),in the 5?-flanking region of the rabbit SP-A gene.In the present study,the PBE was used to screen a rabbit fetal lung cDNA expression library;a cDNA insert was isolated which is highly similar in sequence to hu-man upstream stimulatory factor 1(hUSF1).By use of reverse transcription polymerase chain reaction,two isoforms of rabbit USF1(rUSF1)mRNAs were identified in fetal rabbit lung and other tissues.The levels of rUSF1mRNAs reach a peak in fetal rabbit lung at 23days gestation,in concert with the time of initiation of SP-A gene transcription.Binding complexes of nuclear proteins obtained from fetal rabbit lung tissue and iso-lated type II cells with the DBE and PBE were super-shifted by the addition of anti-rUSF1IgG.Binding activ-ity was enriched in type II cells compared with lung fibroblasts.Overexpression of rUSF1s in A549adenocar-cinoma cells positively regulated SP-A promoter activ-ity of cotransfected reporter gene constructs.It is sug-gested that rUSF1s,which bind to two E-box elements in the SP-A gene 5?-flanking region,may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells.

Surfactant,a developmentally regulated lipoprotein pro-duced by pulmonary type II cells,acts to reduce alveolar sur-face tension and prevent atelectasis;surfactant production is initiated in fetal lung only after ?75%of gestation is com-pleted.The adaptation of the fetus to extrauterine life is highly dependent upon the maturity of the lung at birth and its capacity to produce surfactant.Lung surfactant contains at least four associated proteins,surfactant protein (SP)1-A,

SP-B,SP-C,and SP-D,which appear to serve important roles in surface activity,phospholipid reutilization,and immune function within the alveolus (1).The surfactant protein genes are developmentally regulated and expressed in a lung-specific manner.The gene encoding SP-A is expressed primarily in type II cells and to a lesser extent in bronchioalveolar epithelial (Clara)cells (2,3).SP-A gene expression in fetal lung is under multifactorial control;glucocorticoids and agents that increase cyclic AMP appear to play important roles in its regulation (4).Transcription of the SP-A gene is initiated in fetal rabbit lung tissue on day 24of a 31-day gestation period.Within the 5?-flanking region of the rabbit SP-A gene,we have identified two E-box-like motifs,termed distal binding element (DBE at ?985bp)and proximal binding element (PBE at ?85bp),which bind rabbit lung nuclear proteins in an apparently iden-tical manner (5).Binding activity is enriched in type II pneu-monocytes compared with whole lung tissue.The DBE and PBE share a similar sequence and compete for binding to the same size species of nuclear proteins of 69,45,and 22kDa (5).In type II cell transfection studies,we found that rabbit SP-A gene 5?-flanking sequences between ?991and ?47bp mediate maximal levels of basal and cyclic AMP-induced expression of SP-A promoter activity (6).The finding that mutagenesis of either of the E-boxes (5)or a cyclic AMP-response element (CRE)-like sequence (6,7)resulted in a marked decrease of basal and cyclic AMP-induced expression of SP-A:hGH fusion genes in transfected type II cells indicates that these elements act in a cooperative manner to regulate SP-A promoter activity.In the present study,we screened a rabbit fetal lung cDNA expression library using the PBE sequence as probe.A cDNA insert was isolated encoding the rabbit homolog of human USF1.By use of reverse transcription-polymerase chain reac-tion (RT-PCR)we found that there are two alternatively spliced forms of USF1mRNA in rabbit tissues.Rabbit USF1s (rUSF1s),which are enriched in type II cells,bind to the DBE and PBE and positively regulate SP-A promoter activity.The finding that the levels of rUSF1mRNA reach a maximum in fetal rabbit lung just before the time of initiation of SP-A gene transcription suggests that this transcription factor may play a role in the developmental regulation of SP-A gene expression.

EXPERIMENTAL PROCEDURES

Cloning of a cDNA Insert Encoding a PBE-binding Protein—First strand cDNA was synthesized from poly(A)?RNA isolated from 24-day fetal rabbit lung tissues using random hexanucleotides and was used to generate second strand cDNA using a cDNA synthesis kit (Pharmacia Biotech Inc.,You-Prime cDNA synthesis kit).The double-stranded cDNAs with Eco RI/Not I linkers were inserted into a ?gt11vector and packaged by use of Gigapack II gold packaging extract (Stratagene).A

*This research was supported in part by Grant R01HL50022from the National Institutes of Health (to C.R.M.)and by Grant-in-aid 94R-084from the American Heart Association Texas Affiliate,Inc.(to E.G.).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.

The nucleotide sequence(s)reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s)AF003894for rUSF1a and AF003895for rUSF1b.

?To whom correspondence should be addressed:Dept.of Biochemis-try,The University of Texas Southwestern Medical Center at Dallas,5323Harry Hines Blvd.,Dallas,TX 75235-9038.Tel.:214-648-2944;Fax:214-648-8856;E-mail:cmende@https://www.sodocs.net/doc/aa16944793.html,.1

The abbreviations used are:SP,surfactant protein;AD,activation domain;bHLH-LZ,basic helix-loop-helix-leucine zipper;bp,base pair(s);CIS,competitive internal standard;CMV,cytomegalovirus;CRE,cyclic AMP response element;CREB,CRE-binding protein;DBD,DNA binding domain;DBE,distal binding element;EMSA,electro-

phoretic mobility shift assay;GST,glutathione S -transferase;hGH,human growth hormone;hUSF1,human upstream stimulatory factor 1;PBE,proximal binding element;PCR,polymerase chain reaction;RT,reverse transcription;rUSF1,rabbit upstream stimulatory factor 1.

T HE J OURNAL OF B IOLOGICAL C HEMISTRY

Vol.272,No.37,Issue of September 12,pp.23398–23406,1997

?1997by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.

This paper is available on line at https://www.sodocs.net/doc/aa16944793.html,

23398

by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

32

P-labeled double-stranded oligonucleotide corresponding to the PBE was used to screen the ?gt11cDNA expression library employing stand-ard techniques (8).Upon screening of ?2million recombinant phage clones,a specific cDNA insert encoding a protein that bound specifically to the radiolabeled DBE and PBE,but not to nonspecific DNA,was isolated.The cDNA insert was subcloned into the pGEM-7Z plasmid vector (Promega)and sequenced using Sequenase 2.0(U.S.Biochemi-cal Corp.).The nucleic acid sequence of the 950-bp cDNA insert,termed pG-U1b,was found to be highly similar to the sequence of human upstream stimulatory factor 1(hUSF1)but lacked a 5?-untranslated region and 18bp of sequence coding the amino terminus of the protein.This insert was labeled with 32P and used to rescreen the cDNA library.Another cDNA clone of 1,566bp in length,which contains 126bp of 5?-untranslated region,the full-length coding region,and 594bp of 3?-untranslated region,was isolated,subcloned into pGEM-7Z plasmid vector (termed pG-rUSF1b),and sequenced.

RT-PCR—Poly(A)?RNA isolated from 28-day fetal rabbit lung tissue was annealed to primer 3(Fig.1B and Table I)at 65°C for 5min,and the first strand cDNAs were synthesized by RT using a first strand cDNA synthesis kit (Pharmacia).The synthesized first strand cDNAs were amplified using primers 1and 2(Fig.1B and Table I)by PCR,and plasmid pG-U1b was used as a template in a parallel control reaction.The PCR products were resolved on a 1.8%agarose gel.The DNAs in two bands on the gel were isolated;the DNA isolated from the upper band (termed fragment U1a)was amplified again by PCR.The two isolated RT-PCR products were sequenced by use of CircumVent ther-mal cycle sequencing kit (New England BioLabs).

For quantitative RT-PCR,a DNA fragment to be used as competitive internal standard (CIS)was generated by PCR amplification of the region from 275to 545bp of the plasmid pGEM-7Z (Promega)with a pair of primers that contain the sequences of primers 1and 4at their 5?-ends,which hybridize to the 303–324and 932–949nucleotide se-quences in rUSF1a cDNA (Fig.1A and Table I),respectively.A first strand cDNA synthesis kit was used with 50?g of total RNA isolated from lung tissues of fetal rabbits of 21,23,25,and 28days gestation as templates for reverse transcription of rUSF1cDNAs using the primer 5(Fig.1B and Table I),which hybridizes to the 967–987nucleotide region of rUSF1b mRNA.The cDNAs were washed four times with TE buffer (pH 8.0)using Microcon-100(Amicon)to remove free primers that were not incorporated into the cDNAs.The cDNAs (transcribed from 2.5?g of RNA)were then combined with 1amol (10?18mol)of CIS and used as the templates for PCR,using the 32P-labeled primers 1and 4(Fig.1A and Table I).After 25cycles of amplification,aliquots (40%of products)were resolved on a 1.8%EtBr-agarose gel.The gel was dried,and an autoradiogram was generated and scanned using a 300-A computing laser densitometer.To monitor RT efficiency,0.2?g of oligo(dT)primer (provided by Pharmacia)and 10?Ci of [?-32P]dCTP were used in the parallel reaction.The rate of [?-32P]dCTP incorporation was deter-mined by measurement of radioactivity adsorbed to DE81filters (9).Construction of Plasmids (Table II)—To construct plasmid pG-rUSF1a,the RT-PCR product U1a containing the region of rUSF1a spliced out of rUSF1b was digested with Hin cII and Eco 47III,gel purified,and used to replace the region between Hin cII and Eco 47III of USF1b in pG-rUSF1b (Fig.1).To construct the expression plasmid pQE-U1b (2–161),the region corresponding to amino acids 2–161in pG-rUSF1b was amplified by PCR,and Bam HI and Sal I sites were introduced at the 5?-and 3?-ends,respectively.The PCR-amplified fragment cut with Bam HI and Sal I was subcloned into pQE-30(Qiagen).

For in vivo binding studies using a yeast system,two cDNA frag-ments of rUSF1b were linked either to the Gal4DNA binding domain (in yeast expression vector pGBT9from CLONTECH),referred to as GalDBD or the Gal4activation domain (in yeast expression vector pGAD424from CLONTECH),referred to as GalAD.A cDNA fragment,termed U1b bHLH-LZ ,which encodes the region from the bHLH-leucine zipper region to the stop codon (amino acid residues 127–282),was amplified from pG-U1b by PCR;Eco RI and Bam HI sites were intro-duced at the 5?-and 3?-ends,respectively.The PCR-amplified fragment cut with Eco RI and Bam HI was subcloned into the pGBT9and pGAD424to produce fusion constructs with sequences encoding either the Gal4DNA binding or the transcription activation domains,pDBD-U1b bHLH-LZ and pAD-U1b bHLH-LZ ,respectively.The other PCR-gener-ated cDNA fragment,termed U1b full ,which encodes the region from amino acid 7to the stop codon (amino acid residues 7–282)(Fig.1A ),was used to construct pDBD-U1b full and pAD-U1b full .The yeast expres-sion vector pGAL4,which encodes full-length GAL4protein,was ob-tained from CLONTECH in the Matchmaker Two-hybrid System.To construct pTS381,an Eco RI/Hin dIII fragment of the rabbit SP-A 5?-flanking sequence from ?381to ?49bp (5)was ligated into the Eco

RI

F I

G .1.Rabbit lung contains two isoforms of USF1.Panel A ,schematic diagrams of coding regions of USF1cDNAs.Shown are hUSF1,U1b,isolated by screening the ?gt11rabbit lung cDNA library with 32P-labeled PBE,full-length rUSF1,both of which lack 28amino acid residues corresponding to transactivation domain (TA)II of hUSF1(indicated by the bent lines ),as well as another isoform of USF1in rabbit lung,rUSF1a,obtained by RT-PCR,which contains the 28-amino acid residue TA II present in hUSF1.rUSF1a and U1b cDNAs lack sequences encoding amino acid residues 1–6.The numbers above the lines indicate numbers of the deduced amino acid residues,and those below the lines indicate nucleotide numbers of the cDNA with the translation start site of rUSF1b numbered as 1.The regions to which RT-PCR primers 1,4,and 5anneal are indicated by arrows below the line representing rUSF1a cDNA.

H ,Hin cI

I site;E ,Eco 47III site;B ,Bgl II site.Primers used in RT-PCR are indicated.Primer 3was used to synthesize the first strand cDNA from day 28fetal rabbit lung RNA by RT.The cDNA was amplified by PCR using two other oligonucleotides,primers 1and 2.Panel B ,RT-PCR products of two isoforms of rUSF1s resolved by agarose gel https://www.sodocs.net/doc/aa16944793.html,ne 1,rUSF1b cDNA (clone U1b)was used as template in PCR as a control;lane 2,products of RT-PCR of day 28fetal rabbit lung RNA.

T ABLE I

Oligonucleotides used

Name

Sequence (5?to 3?)

Primer 1TGATACGGTTGACACAGAGGGGACA Primer 2GGGCTCATGAGCGCTGGCTTCCCCCCTG Primer 3GGGCCGCTGACTTCGGGGAAT Primer 4CCCCCACATCCCCATGGT Primer 5

CTGTTGCTCCTGGGCTAGCTG DBE GATCTCC CACGTG GGTGCAGGG a PBE GAGGCC CTCGTG ACAGGG b

D-m1GATCTCC T ACGTG GGTGCAGGG c D-m2GATCTCC CA TA TG GGTGCAGGG c P-m1GAGGCC T TCGTG ACAGGG c P-m2GAGGCC CT TA TG ACAGGG c

AdML GTGTAGGC CACGTG ACCGGGTGTT a CRE

CTCCTTGGC TGACGTCA GAGAGAG d

a

The E-box sequence,CACGTG,of the rabbit SP-A gene (DBE)and adenovirus major late (ADML)promoter is shown in bold letters.b

The E-box-like sequence,CTCGTG,of the rabbit SP-A gene (PBE)is shown in bold letters.c

The mutated nucleotides in the E-box are shown in italics and underlined.d

An 8-bp palindromic cyclic AMP-responsive element that binds to transcription factor CREB (13)is shown in bold letters.

Transcriptional Regulation of SP-A by USF1

23399

by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

site of pTH1(a kind gift from Dr.S.Fields),filled in,and ligated.

For transfection studies,SP-A:hGH fusion genes SP-A ?991:hGH,SP-A ?976:hGH,SP-A ?991PBE(?):hGH,containing ?991,?976(lacking the DBE),and ?991(containing a mutation in the PBE)bp of 5?-flanking DNA from the rabbit SP-A gene linked to the human growth hormone (hGH)structural gene (5,6),and expression plasmids pACSKCMV2and pCMV-nLac were utilized.An adaptor containing Nsi I and Eco RV sites followed by the translation start codon ATG and an Eco RI site was inserted into the Eco RI site of pACSKCMV2to generate vector pCMV.pCMV-USF1a and pCMV-USF1b were constructed by subcloning Eco RI fragments of pG-rUSF1a and pG-U1b into pCMV,respectively.

pGST-U1b,used for expression of the glutathione S -transferase (GST)-rUSF1b fusion protein in bacteria,was constructed by cloning the Eco RI fragment of pG-U1b into the Eco RI sites of pGEX-1?T (Pharmacia).

The orientations and the sequences of linking sites were confirmed by DNA sequencing.In all cases where inserts were generated by PCR,the nucleotide sequences in the resulting plasmid inserts were con-firmed by DNA sequencing.

Expression and Purification of GST and His-tag Fusion Proteins—GST and GST-fusion proteins were expressed and purified from Esch-erichia coli DH-5?(Life Technologies,Inc.)as described by Smith and Johnson (10).After binding to glutathione-Sepharose 4B (Pharmacia),the proteins were washed and eluted with reduced glutathione (Sigma).His-tag fusion proteins were expressed and purified from E.coli M15[pREP4](Qiagen)according to the manufacturer’s instructions.After binding to Ni-NTA-resin (Qiagen),the proteins were washed and eluted with imidazole (Sigma).The concentrations of the expressed proteins were determined by the method of Bradford (Bio-Rad).The purity and size of the eluted proteins were then evaluated by Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels.

Preparation of USF1Antibodies—Polyclonal antibodies to rUSF1were generated by immunizing guinea pigs as described by Harlow and Lane (11)with His-tagged USF1b (2–161).The IgGs were purified by use of an ImmunoPure (A)IgG purification kit (Pierce)according to the manual.This IgG preparation recognized both expressed rUSF1a and

rUSF1b by immunoblot analysis.

Electrophoretic Mobility Shift Assay (EMSA)—Rabbit lung nuclear proteins were prepared as described by Gorski et al.(12).Type II pneumonocytes and fibroblasts were prepared from cultured fetal rab-bit lung explants (6).Binding reactions and gel electrophoresis all were performed as described previously (5).Double-stranded oligonucleo-tides corresponding to the DBE,PBE,and CRE (13)(Table I)were end labeled using polynucleotide kinase and [?-32P]ATP.The nuclear ex-tracts (10?g),bacterially expressed GST (240ng)or GST-rUSF1b fusion proteins (75ng)were incubated at room temperature for 20min in binding buffer (20m M HEPES (pH 7.6),150m M KCl,0.2m M EDTA,20%glycerol)with radiolabeled DNA probe and poly(dI ?dC)-poly(dI ?dC)(Pharmacia)as nonspecific competitor.For antibody supershift analy-sis,this was followed by the addition of guinea pig preimmune IgG or the IgG against rUSF1s (13ng in 1?l)and an additional 20-min incubation.The DNA-protein complexes were resolved on a 5%native polyacrylamide gel and visualized by autoradiography.

In Vivo Binding Analysis in Yeast—The reporter gene-containing plasmid pTS381was linearized by digestion with Pvu II in the LYS2region and transformed into yeast w303-1a (a kind gift from Dr.S.Fields).The pTS381-integrated strain,termed S381,was selected by growing in medim lacking uracil and confirmed by Southern blotting.S381was transformed with pDBD-U1b bHLH-LZ (expresses GalDBD-U1b bHLH-LZ ),pDBD-U1b full (expresses GalDBD-U1b full ),pAD-U1b bHLH-LZ (expresses GalAD-U1b bHLH-LZ ),and pAD-U1b full (expresses GalAD-U1b full ),as well as with control plasmids pGBT9(expresses GalDBD),pGAD424(expresses GalAD),and pGAL4(expresses full-length Gal4protein).A single colony was grown at 30°C for 16h in SD medium lacking tryptophan (?Trp)for the transformants of pGBT9-derived plasmids,or in SD medium lacking leucine (?Leu)for the transformants of pGAD424-derived plasmids (14).The cultures were washed three times with water and diluted to 0.5O.D.600/ml.Depend-ing upon which of the transformed plasmids were being analyzed,4?l of diluted yeast was applied either to plates lacking histidine (?His)and tryptophan (?Trp)or ?His and ?Leu SD/3-aminotriazole (5m M )plates as well as to ?Trp or ?Leu SD plates.The plates were incubated

T ABLE II

Plasmid constructs

Name

Insert/vector

Usage

pG-U1b rUSF1b cDNA a /pGEM-7Z Original cDNA pG-rUSF1b rUSF1b cDNA b /pGEM-7Z Original cDNA pG-rUSF1a

rUSF1a cDNA/pGEM-7Z

Original cDNA pQE-U1b (2-161)cDNA for amino acids 2–161of rUSF1b/pQE-32

Raising antibodies pGBT9Gal4DNA binding domain Expression of GalDBD in yeast pGAD424Gal4activation domain

Expression of GalAD in yeast pGAL4

cDNA encoding full-length Gal4

Expression of Gal4in yeast

pDBD-U1b bHLH-LZ cDNA encoding the bHLH-LZ domain of rUSF1b linked to Gal4DNA binding domain/pGBT9

Expression

of

GalDBD-U1b bHLH-LZ in yeast

pDBD-U1b full cDNA encoding rUSF1b full c linked to Gal4DNA binding domain/pGBT9

Expression of GalDBD-U1b full in yeast pAD-U1b bHLH-LZ cDNA encoding the bHLH-LZ domain of rUSF1b linked to Gal4activation domain/pGAD424

Expression of GalAD-U1b bHLH-LZ in yeast pAD-U1b full cDNA encoding rUSF1b full c linked to Gal4activation domain/pGAD424

Expression of GalAD-U1b full in yeast pTS381Rabbit SP-A 5?-flanking DNA (?381to ?49bp)linked to His3gene/pTH1Reporter gene in yeast pGST-U1b cDNA encoding full-length rUSF1b c /pGEX-1?T

EMSA

SP-A ?991:hGH Rabbit SP-A DNA (?991to ?20bp with the DBE deleted)linked to hGH structural gene/pUC12

Fusion reporter gene SP-A ?976:hGH Rabbit SP-A DNA (?976to ?20bp with the DBE deleted)linked to hGH structural gene/pUC12

Fusion reporter gene

SP-A ?991PBE(?):hGH Rabbit SP-A DNA (?991to ?20bp,with the PBE mutated)linked to hGH structural gene/pUC12Fusion reporter gene pACSKCMV2CMV promoter/pBluescript

pCMV

CMV promoter and start codon ATG/pBluescript Empty expression vector pCMV-nLac cDNA encoding ?-gal d linked to CMV promoter/pBluescript

Transfection efficiency assay pCMV-USF1a CDNA encoding rUSF1a linked to CMV promoter/pBluescript

Expression of rUSF1a pCMV-USF1b

cDNA encoding rUSF1b linked to CMV promoter/pBluescript

Expression of rUSF1b

a Originally isolated from cDNA library using the PBE as probe,lacking 6amino acids at NH 2terminus.b

Isolated from cDNA library,containing 5?-untranslated region.c

rUSF1b full ,the cDNA encoding rUSF1b from amino acid 7to the stop codon.d

?-gal,?-galactosidase.

Transcriptional Regulation of SP-A by USF1

23400 by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

at 30°C for 48h and photographed.To quantify the growth of trans-formants,30?l of diluted yeast was incubated in 5ml of either ?His and ?Trp or ?His and ?Leu SD/3-aminotriazole (5m M )medium at 30°C for 22h.The absorbancies of the cultures at 600nm were determined.

Homodimerization of rUSF1in a Yeast Two-hybrid System—To study the interaction of rUSF1in vivo ,the Gal4DBD-USF1fusion plasmids pDBD-U1b bHLH-LZ and pDBD-U1b full ,the Gal4activation do-main-USF1fusion plasmids pAD-U1b bHLH-LZ and pAD-U1b full ,as well as control plasmids pGBT9,pGAD424,and pGAL4were transformed into yeast HF7c (provided in the Matchmaker two-hybrid system kit,CLONTECH)individually or cotransformed in different combinations.The transformants were grown in appropriate medium and studied as described above.

Transient Transfection of USF1Expression Vectors and Reporter Gene Constructs—A549cells (ATCC CCL 185)(15)were plated at a density of 5–9?106cells/60-mm dish 1day before transfection.The cells were maintained overnight in Waymouth’s MB752/1medium con-taining fetal calf serum (10%,v/v).The cells were then washed three times with Hanks’balance salt solution (Life Technologies,Inc.)and cotransfected with a number of SP-A:hGH reporter gene constructs,USF1expression vectors,and control plasmids by incubation with 11?g of each DNA fragment and 44?g of DOTAP (Boehringer Mannheim)in Waymouth’s MB752/1for 18h.The medium was then aspirated and replaced with fresh Waymouth’s MB752/1at 24-h intervals.Two days later,the media and cells were collected.The concentrations of hGH in the media were analyzed by radioimmunoassay using an Allegro hGH kit (Nichols Institute Diagnostics,San Juan Capistrano,CA).?-Galac-tosidase activity was determined as described by Miller (16).

RESULTS

Isolation of the Rabbit USF1cDNA Clones—To isolate cDNA clones encoding proteins that bind to E-box motifs of the DBE and PBE in the 5?-flanking region of the rabbit SP-A gene,a ?gt11cDNA expression library constructed from mRNA iso-lated from 24-day fetal rabbit lung tissue was screened using a 32

P-labeled double-stranded oligonucleotide corresponding to the PBE.From ?2million recombinant phage clones,a specific cDNA clone encoding a protein that bound specifically to the radiolabeled DBE and PBE (Table I)but not to nonspecific DNA C2(5)was isolated.The cDNA insert was subcloned into the pGEM-7Z plasmid vector and sequenced.The nucleic acid sequence of the 950-bp cDNA insert,termed pG-U1b,was found to be highly similar to the sequence of hUSF1.This clone contained an 828-bp open reading frame and 113bp of 3?-untranslated DNA but lacked 5?-untranslated region and 18bp of coding sequence at the amino terminus of the protein.To isolate a full-length cDNA,the insert in pG-U1b was used to rescreen the cDNA library,and a 1,566-bp cDNA insert was isolated which contained a full-length cDNA for rUSF1b.This clone contained 126bp of 5?-untranslated region,an 846-bp open reading frame,and 594bp of 3?-untranslated region.A comparison of the sequences of hUSF1and rUSF1b revealed that only two amino acid residues of rUSF1b differ from the corresponding sequence of hUSF1;however,rUSF1b lacks 28amino acid residues in transactivation domain II of hUSF1(17).This region is indicated by the angled line in Fig.1A .Rabbit Tissues Contain Two Isoforms of USF1mRNA—To determine whether there is an isoform of USF1in rabbit lung which contains the 28amino acid residues that are lacking in rUSF1b,we designed an oligonucleotide primer (primer 3)which anneals to a region 100bp downstream of the deleted region in the rUSF1b (Fig.1A and Table I).This was used to synthesize,by reverse transcription,the first strand cDNA from 28-day fetal rabbit lung RNA.The cDNA was amplified by PCR using two other oligonucleotide primers,1and 2(Fig.1A and Table I).As shown in lane 2of Fig.1B ,two RT-PCR products were obtained.The major product corresponded in length to rUSF1b (lower band ,154bp),whereas the other corresponded in length to hUSF1(upper band ,238bp,termed rUSF1a).Sequence analysis of the RT-PCR-amplified cDNA

revealed that rUSF1a is 100%identical to rUSF1b except that rUSF1a contains the sequence encoding 28amino acid residues absent in rUSF1b.This 28-amino acid sequence,deduced from the nucleotide sequence of the rUSF1a,is identical to the corresponding region in hUSF1.Interestingly,in RNA isolated from day 28fetal rabbit lung,rUSF1a was found to be present at much lower levels than rUSF1b (Fig.1B ).The two isoforms of rUSF1also were found in other tissues by RT-PCR (below).USF1Comprises One Component of the Complex of Proteins Bound to the DBE and PBE—To obtain expressed rUSF1pro-teins for analysis of their properties,the rUSF1b cDNA insert was subcloned into the GST fusion vector pGEX-1?T (Pharma-cia)for expression and purification of GST-USF1b fusion pro-tein.The GST-rUSF1b fusion proteins expressed in E.coli were purified using glutathione-agarose beads and used to analyze binding activity for the DBE and PBE by EMSA.As shown in Fig.2,28-day fetal rabbit lung nuclear proteins (lanes 6,9,and 12)and GST-rUSF1b (lanes 5,8,and 11)bound to the radiola-beled DBE (upper panel )and PBE (lower panel ).When in vitro translated rUSF1b,which does not contain GST,was used in EMSA,the same mobility shift complexes were observed (data not shown).Although GST-rUSF1b fusion protein is about 26kDa larger than rUSF1b,its binding complexes with the DBE or PBE displayed an electrophoretic mobility similar to that of native rUSF1-containing complexes (lanes 5versus 6),suggest-ing that folding and/or net charge of rUSF1plays a more important role in mobility in native polyacrylamide gels than molecular weight.The binding complexes of the DBE and PBE with fetal rabbit lung nuclear proteins were partially super-shifted by the addition of anti-rUSF1IgG (lane 12),indicating that one of the nuclear proteins that bound to the DBE and PBE is USF1.The anti-rUSF1IgG completely supershifted the complexes of expressed GST-rUSF1b bound to the DBE and PBE (lane 11

).

F I

G https://www.sodocs.net/doc/aa16944793.html,F1s comprise one component of the complex of pro-teins bound to the DBE and PBE.Radiolabeled DBE (upper panel )or PBE (lower panel )was incubated for 20min with bacterially ex-pressed GST,GST-rUSF1b fusion protein,or with nuclear proteins isolated from lung tissues of 28-day fetal rabbits,followed by incubation with anti-rUSF1IgG or with preimmune guinea pig IgG,as control,for 20min before electrophoresis.F ,free probe;C ,DNA-protein complexes;S ,supershift of DNA-protein-binding complexes with anti-rUSF1IgG.

Transcriptional Regulation of SP-A by USF1

23401

by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

Although the sequence of the PBE (CTCGTG)differs by one nucleotide from the DBE E-box (CACGTG),which contains a sequence known to bind to hUSF1(18),the PBE has binding activity similar to the DBE for expressed rUSF1b (Fig.3)and rabbit lung nuclear proteins (5).The PBE and the DBE com-pete for binding to lung nuclear proteins (5).This may be explained by the finding that the nucleotide residues C and G are the most important nucleotides for binding (18).To analyze the importance of the nucleotide sequences of the DBE and PBE for rabbit lung nuclear protein binding,a series of oligo-nucleotides containing one (m1)or two (m2)point mutations at the known contact points (Table I)were generated and utilized in competition EMSA using the wild type DBE and PBE se-quences as radiolabeled probes.As shown in Fig.3,the binding of radiolabeled DBE either to bacterially expressed GST-rUSF1b or to lung nuclear proteins was effectively competed by a 500-fold molar excess of nonradiolabeled DBE or of a 24-bp oligonucleotide from the adenovirus major late promoter (ADML)(19)which contains the E-box core sequence,CACGTG,but a different flanking sequence (Table I).The ability to compete with DBE for binding to GST-rUSF1b and nuclear proteins was abolished when the 5?C in the E-box core sequence of the DBE was mutated to T (D-m1).A nonradiola-beled oligonucleotide in which the central C and G of the E-box core sequence of the DBE were mutated to T and A (D-m2,leaving the E-box consensus sequence,CANNTG,intact)also was an ineffective competitor of the radiolabeled DBE at a 500-fold molar excess (Fig.3).When the DBE mutants were used in EMSA as radiolabeled probes,only D-m2,but not D-m1,bound GST-rUSF1b and lung nuclear proteins but with

a very low activity (data not shown).The same results were obtained when the competition experiment was performed us-ing radiolabeled PBE as probe and PBE mutants as competi-tors (Fig.3).

The SP-A gene is expressed predominantly in type II pneu-monocytes and to a lesser extent in Clara cells of rabbit lung tissue (2).Binding activity of rabbit fetal lung nuclear proteins for the DBE and PBE is enriched in type II cells compared with whole lung tissue (5).To evaluate the possible role of rUSF1in the type II cell-specific regulation of SP-A gene expression,the binding activity of rUSF1in nuclear proteins from an enriched population of the type II cells was compared with that of nu-clear proteins from an enriched population of fibroblasts iso-lated from the same collagenase-digested cell suspension of cultured fetal rabbit lung tissue using an electrophoretic mo-bility supershift assay and radiolabeled DBE and PBE.As can be seen in Fig.4,binding activity for radiolabeled DBE and PBE was greatly enriched in type II cells (lane T II )compared with lung fibroblasts (lane Fb ).The binding complexes of the DBE and PBE with fetal rabbit lung nuclear proteins were partially supershifted by the addition of anti-rUSF1IgG,indi-cating that one of the type II cell nuclear proteins that bound to the DBE and PBE was USF1.To evaluate the integrity of the fibroblast and type II cell nuclear extracts,we compared their abilities to bind a radiolabeled double-stranded oligonucleotide containing the CRE sequence,TGACGTCA,derived from the rat somatostatin gene (Table I),which binds to the ubiquitous transcription factor CREB (13).Binding activities of nuclear proteins from fibroblast and type II cells for the CRE were similar (data not shown),indicating that these two prepara-tions contained equivalent amounts of nuclear protein binding activity.

USF1Homodimerizes in Vivo—It has been found that hUSF1binds to the E-box motif as a dimer and that both helix-loop-helix and leucine zipper domains are required for dimerization (17,20,21).By studying the interaction of 35S-labeled rUSF1b and bacterially expressed GST-USF1b fusion proteins in the presence or absence of the DBE and PBE

in

F I

G .3.Analysis of the effects of mutation of the DBE and PBE on binding of GST-rUSF1b or 28-day fetal rabbit lung nuclear proteins using competitive EMSA.Radiolabeled DBE (left panels )or PBE (right panels )was incubated for 30min with bacterially ex-pressed GST-rUSF1b fusion protein (upper panels )and with nuclear proteins isolated from lung tissues of 28-day fetal rabbits (lower panels )in the absence (?)or presence of double-stranded oligonucleotides con-taining DBE or PBE without or with one (m1)or two (m2)mutations in the E-box core sequence or ADML as competitors at 500-fold molar excess of the radiolabeled probe.The mutations in the E-box core sequence are shown in bold italics

.

F I

G .4.rUSF1binding activity for the DBE and PBE is en-riched in lung type II cells.Radiolabeled DBE (upper panel )or PBE (lower panel )was incubated with nuclear proteins isolated from fetal rabbit lung fibroblasts (Fb )or type II cells (T II )for 20min followed by incubation for 20min in the absence or presence of anti-rUSF1IgG,followed by electrophoresis.F ,free probe;C ,DNA-protein complexes;S ,antibody supershift of DNA-protein complexes with anti-USF1IgG.

Transcriptional Regulation of SP-A by USF1

23402 by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

vitro ,we found that rabbit USF1also can form homodimers in solution before binding to the specific E-box elements and that the binding of rUSF1to the E-box does not influence dimeriza-tion of USF1(data not shown).The yeast two-hybrid system (22)was used to determine whether homodimerization of rUSF1occurs in vivo and whether the presence of the amino-terminal region compared with bHLH-LZ alone influences dimerization.As shown in Fig.5,both the bHLH-LZ region of rUSF1(U1b bHLH-LZ )and full-length rUSF1b (U1b full )ho-modimerized and transactivated expression of the His3gene in yeast HF7c.The efficiency of the bHLH-LZ region for interac-tion was only one-third of that of the full-length rUSF1b.This could possibly be the result of a fusion protein artifact,since the bHLH-LZ region contains all essential domains for DNA binding and homodimerization (21).

The Levels of rUSF1a and rUSF1b mRNA in Rabbit Lung Tissue Change during Fetal and Postnatal Development—SP-A gene expression is developmentally regulated in fetal lung tis-sue (4).In rabbits,SP-A gene transcriptional activity is first detectable on day 24and reaches maximal levels by day 28of the 31-day gestation period (24).To analyze developmental changes in the levels of rUSF1mRNAs,quantitative RT-PCR experiments were performed.First,we developed a CIS,a nonhomologous DNA fragment from the plasmid pGEM-7Z engineered to contain the rUSF1primer templates (the regions

corresponding to nucleotides 303–324and 932–949in rUSF1a cDNA,Fig.1A )at 5?-and 3?-ends,respectively.The CIS was of a size to generate a CIS PCR product of 311bp in length.50?g of total RNA isolated from day 28fetal rabbit lung was used as template for reverse transcription of rUSF mRNAs using the primer 5that hybridizes to nucleotides 967–987of rUSF1a mRNA (Fig.1A ).The cDNAs that were generated were then used as the templates for PCR,using the 32P-labeled primers 1and 4that hybridize to nucleotides 304–323and 932–949of rUSF1cDNA (Fig.1A ).These primers flank the segment en-coding the 28amino acid residues that are deleted in rUSF1b;therefore,the cDNAs specific for both rUSF1a and rUSF1b can be amplified as 557-bp and 641-bp products,respectively.An aliquot of cDNAs corresponding to reverse transcripts from 2.5?g of RNA were used in each reaction in the presence of varying amounts of CIS.After 25cycles of amplification,PCR product accumulation remained exponential so that product concentra-tion was proportional to starting cDNAs.Aliquots of products (40%of total)were resolved on an agarose gel and visualized by autoradiography (upper panel of Fig.6A ).The majority of rUSF1RT-PCR products was comprised of rUSF1b (557bp),whereas rUSF1a (641bp)constituted only a minor component.Under competitive conditions,the amount of rUSF cDNA syn-thesized is equivalent to the amount of CIS added to the reac-tion when the molar ratio of products ?1.The point at which CIS and rUSF1b products were equal was reached at 2.5?10?19mol of CIS (lower panel of Fig.6B ),suggesting that there are about 10?19mol of rUSF1b mRNA/?g of total RNA in 28-day fetal rabbit lung.

To analyze developmental changes in the levels of rUSF1mRNAs,aliquots of total RNA isolated from lung tissues of 21–28-day fetal rabbits and from neonates were used as tem-plates to synthesize first strand cDNAs of rUSF1by reverse transcription as described above.The efficiency of reverse tran-scription was monitored by use of oligo(dT)primer and [?-32P]dCTP in a parallel reaction.The percentage of [?-32P]dCTP incorporation was 1.22?0.17(mean ?S.D.).PCR was carried out using the cDNA transcribed from 2.5?g of RNA in the presence of 1amol (10?18mole)of CIS.As shown in the autoradiogram (upper panel of Fig.6B ),the majority of rUSF1RT-PCR products was comprised of rUSF1b (557bp),whereas rUSF1a (641bp)consisted of only a minor component at all developmental ages studied.The levels of rUSF1mRNAs reached a peak at 23days (lower panel of Fig.6B ),which is just prior to the time that initiation of SP-A gene transcription can be detected in nuclei from fetal rabbit lung (3).Interestingly,the ratio of rUSF1a to rUSF1b also reached a maximal level at this time.

mRNA Encoding rUSF1s Are Expressed Ubiquitously in Fe-tal Rabbit Tissues—USF1is a ubiquitously expressed tran-scription factor (23).To determine whether USF1exists in two isoforms in different tissues,aliquots of total RNA isolated from heart,skeletal muscle,liver,kidney,and brain of 23-day fetal rabbits were analyzed by RT-PCR using the same USF1primers as in the quantitative RT-PCR experiments.As shown in Fig.7,the same two RT-PCR products that were amplified from lung were isolated using RNA from all of these tissues;the major band corresponds in length to rUSF1b (557bp),whereas the other corresponds in length to rUSF1a (641bp).The levels of rUSF1a mRNA appeared to be highest in muscle and liver and lowest in kidney and brain.On the other hand,rUSF1b signals appeared similar in all tissues examined.This finding suggests that although expression of the USF1gene is equiv-alent in these tissues,alternative splicing of the mRNA may be regulated by tissue-specific factors.Alternatively,the apparent equivalence of the USF1b signal could be the result of

its

F I

G https://www.sodocs.net/doc/aa16944793.html,F1interacts in vivo in a yeast two-hybrid system.Panel A ,plasmids expressing the Gal4DNA binding domain (GalDBD),Gal4activation domain (GalAD),full-length Gal4protein (GAL4),GalDBD fused to the basic-helix-loop-helix domain of rUSF1b (GalDBD-U1b bHLH-LZ ),GalDBD fused to the cDNA encoding rUSF1b from amino acid 7to the stop codon (GalDBD-U1b full ),GalAD fused to the basic-helix-loop-helix domain of rUSF1b (GalAD-U1b bHLH-LZ ),and GalAD fused to the cDNA encoding rUSF1b from amino acid 7to the stop codon (GalAD-U1b full )(Table II)were transformed alone or in the combinations indicated into yeast HF7c.The yeast transformants were grown on a ?His plate containing 5m M 3-aminotriazole for 20h.Panel B ,densities at 600nm were determined as described under “Experi-mental Procedures.”Data are mean ?S.E.of three determinations from one of two similar independent experiments.

Transcriptional Regulation of SP-A by USF123403

by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

greater abundance,causing the number of PCR cycles used to exceed the linear phase of the reaction.

USF1Interaction with an E-box Element within 5?-Flanking Region of the SP-A Gene in Yeast Results in Activation of Yeast Gal1Promoter Activity—To determine whether the binding of rUSF1to the PBE E-box motif transactivates gene expression in vivo ,we used a yeast one-hybrid system.Plasmids pAD-U1b full and pAD-U1b bHLH-LZ ,which express GalAD-U1b full and GalAD-U1b bHLH-LZ ,respectively,were transformed into the yeast S381,in which ?381to ?49bp of 5?-flanking region of the rabbit SP-A gene linked to a minimal Gal1promoter-con-trolled His3gene was incorporated into the yeast genome.The ?381to ?49bp 5?-flanking region contains the functional PBE,and this region has been shown to mediate basal and cAMP-inducible activation of SP-A promoter activity in type II cells (5).Yeast expression plasmids pDBD-U1b full and pDBD-U1b bHLH-LZ ,which express GalDBD-U1b full and GalDBD-U1bHLH-LZ ,respectively,as well as the control plasmids pGAD424,pGBT9,and pGAL4,which express GalAD,GalDBD,and full-length Gal4protein,respectively,also were transformed into yeast S381.As shown in Fig.8,both full-length USF1b and the bHLH-LZ region of USF1linked to the Gal4activation domain (GalAD-U1b full and GalAD-U1b bHLH-LZ ,re-spectively)activated expression of the His3gene in yeast S381,resulting in its ability to grow in medium lacking histidine (?His).By contrast,neither GalDBD,GalAD,full-length Gal4protein (GAL4),nor GalDBD linked either to full-length USF1b (GalDBD-U1b full )or to the USF1b bHLH-LZ region (GalDBD-U1b bHLH-LZ )activated expression of the His3gene in yeast S381.These findings suggest that the rUSF1bHLH domain functionally interacted with the E-box element within the 5?-flanking region of rabbit SP-A gene so that the fused Gal4activation domain activates Gal1promoter activity and expres-

sion of the His3gene.No apparent difference in efficiency for activation of His3gene expression was observed between the full-length and bHLH-LZ region of rUSF1(Fig.8),although full-length USF1exhibited a greater ability to form ho-modimers than the bHLH domain (Fig.5).

USF1Activates Expression of SP-A:hGH Fusion Genes in A549Cells—In previous studies,we found that SP-A:hGH fu-sion genes containing ?991bp of SP-A 5?-flanking sequence (SP-A ?991:hGH)linked to the hGH structural gene were ex-pressed in primary cultures of type II cells;expression of

SP-

F I

G .7.rUSF1a and rUSF1b mRNAs are expressed in different tissues of 23-day gestational fetal rabbits.Aliquots of total RNA isolated from different tissues of fetal rabbits of 23days gestational age were used as templates for reverse transcription of rUSF cDNAs using the primer described under “Experimental Procedures.”The cDNAs were amplified by PCR in the presence of CIS template at 10?19mol/?g of RNA,a nonhomologous DNA fragment containing the rUSF1primer templates which yields a PCR product of 311bp in length.Aliquots of PCR products were resolved on an agarose gel.The gel was dried,and an autoradiogram was

generated.

F I

G .6.The levels of rUSF1a and rUSF1b mRNAs in rabbit lung tissue change during fetal and postnatal development.Panel A ,total RNA isolated from lung tissues of fetal rabbits of 28days gestational age was used as template for reverse transcription of rUSF cDNAs using the primer described under “Experimental Procedures.”The cDNAs were amplified by PCR in the presence of different amounts of CIS,a nonho-mologous DNA fragment containing the rUSF1primer templates which yields a PCR product of 311bp in length.Aliquots of PCR products were resolved on an agarose gel.The gel was dried,and an autoradiogram was generated (upper panel )and scanned using a computing laser densitometer.Shown in the lower panel is the scan of rUSF1b and CIS arbitrary units.Panel B ,total RNAs isolated from lung tissues of fetal rabbits of 21–28days gestational age and from neonates (N )were used as templates for reverse transcription of rUSF1cDNAs.The cDNAs were amplified by PCR in the presence of CIS template at 10?19mol/?g of RNA.Aliquots of the PCR products were resolved on an agarose gel.The gel was dried,and an autoradiogram (upper panel )was generated and scanned using a computing laser densitometer (lower panel ).C ,control in which the RNA was subjected to PCR without prior incubation with reverse transcriptase.Data shown are from one of five similar experiments.

Transcriptional Regulation of SP-A by USF1

23404 by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

A ?991:hGH was stimulated upon incubation with Bt 2cAMP.Mutagenesis of the DBE or PBE in the context of the 991bp of 5?-flanking region resulted in a marked reduction of basal and cyclic AMP-induced fusion gene expression (5).To determine whether rUSF1a and rUSF1b regulate expression of the SP-A gene via the DBE and PBE,expression vector plasmids pCMV-USF1a and pCMV-USF1b were cotransfected into A549cells with the SP-A ?991:hGH fusion gene,with SP-A ?976:hGH (which lacks the DBE)or with SP-A ?991PBE(?):hGH (containing a mutation in the PBE)(5).A549is a lung adenocarcinoma-derived cell line presumed to be of type II cell origin;however,this cell line does not produce SP-A mRNA and protein at detectable levels (15).As shown by the open bars in Fig.9,when A549cells were cotransfected with the intact SP-A ?991:hGH fusion gene and with pCMV-USF1a or pCMV-USF1b,a 3.1-and 2.4-fold induction,respectively,of hGH expression was found compared with that observed upon cotransfection of the SP-A ?991:hGH fusion gene with “empty”expression vector plasmid,pCMV,or calf thymus DNA shown as control.By contrast,when A549cells were cotransfected with pCMV-USF1a or pCMV-USF1b and the SP-A ?976:hGH fusion gene,in which the DBE is deleted (hatched bars ),no induction of hGH expression was found compared with that observed upon co-transfection with the empty vector plasmid pCMV or calf thy-mus DNA (Fig.9).Similarly,no induction of hGH expression was detected when A549cells were cotransfected with SP-A ?991PBE(?):hGH fusion genes and pCMV-USF1a or pCMV-USF1b.When A549cells were cotransfected with SP-A ?991PBE(?):hGH fusion genes and calf thymus DNA or pCMV,hGH expression also was below detectable levels (Fig.9).These findings suggest that rUSF1s bind to the E-box motifs in the 5?-flanking region of the rabbit SP-A gene and induce SP-A promoter activity.

DISCUSSION

Previously,we identified two structurally similar sequences within the 5?-flanking region of the rabbit SP-A gene,termed

DBE and PBE (5).Deletion or mutation of these elements reduces both basal and cAMP-induced expression of SP-A:hGH fusion genes transfected into type II cells.The DBE contains the E-box core sequence CACGTG,which is known to interact with a number of gene regulatory proteins that contain a bHLH structure.The PBE contains the sequence CTCGTG,which differs by one nucleotide from the DBE (5).Because the PBE and the DBE compete for binding to lung nuclear proteins (5)we considered it likely that similar or identical proteins bind to these elements.In the present study,we screened a fetal rabbit lung cDNA expression library using the radiolabeled PBE as probe.A cDNA insert was isolated which encodes a protein that specifically binds to the DBE and PBE elements.Sequence analysis of the cDNA insert revealed that this protein is the rabbit homolog of hUSF1(18,19,25–29),a heat-stable protein,M r Х43,000,which binds to the E-box element CACGTG (26).Previously,we observed that heat-stable type II cell nuclear proteins of M r ?69,000,45,000,and 22,000bind both to the DBE and PBE;the 45-kDa protein(s)appears to be the pre-dominant species and binds as a dimer (5).In the present study,we observed that anti-rUSF1IgG supershifted a major portion of the binding complexes formed upon incubation of nuclear proteins from rabbit lung type II cells with the radio-labeled DBE and PBE.These findings suggest that USF1com-prises a principal component of the complex of lung type II cell nuclear proteins that bind to these elements.

The cDNA isolated in the screen of the rabbit lung cDNA library encodes rUSF1b,a human USF1(hUSF1)homolog that lacks 28amino acids in the second transactivation domain.By use of RT-PCR,we also identified in all tissues analyzed an-other mRNA species for USF1,which we termed rUSF1a,en-coding the full-length 310-amino acid protein;however,we observed that rUSF1b is present at considerably higher levels than rUSF1a in all fetal rabbit tissues studied.Three forms of hUSF1cDNA were characterized previously by Gregor et al.(26),termed z18,z32,and z6b.Only z6b encodes the full-length USF1protein of 310amino acids;z18and z32encode truncated 244-and 49-amino acid isoforms,respectively.Interestingly,none of these forms of hUSF1cDNAs corresponds to

rUSF1b,

F I

G https://www.sodocs.net/doc/aa16944793.html,F1binds to the 5?-flanking region of the rabbit SP-A gene in vivo in yeast.The plasmids that express the fusion proteins indicated in the figure and described in Table II were transformed into yeast S381in which the 5?-flanking region of the rabbit SP-A gene (?381to ?49bp)linked to a minimal Gal1promoter-controlled His3gene had been incorporated into the yeast genome.The yeast transfor-mants were grown on a ?His plate for 20h.Growth was observed only in yeast transformed with GalAD-U1b bHLH-LZ and GalAD-U1b full .Den-sities at 600nm were determined as described under “Experimental Procedures.”Data are the mean ?S.E.of three determinations from one of two similar independent

experiments.

F I

G .9.Expression of SP-A:hG

H fusion genes in A549cells is increased by cotransfection of rUSF1s.The fusion genes SP-A ?991:hGH,SP-A ?976:hGH (DBE is deleted),or SP-A ?991PBE(?):hGH (PBE is mutated)were cotransfected with either calf thymus DNA,pCMV,pCMV-USF1a,or pCMV-USF1b into A549cells.Shown are the levels of immunoreactive hGH that accumulated over a 24-h period.When A549cells were cotransfected with the SP-A ?991PBE(?):hGH fusion gene and calf thymus DNA,pCMV,pCMV-USF1a,or pCMV-USF1b,hGH ex-pression was below detectable levels.Data are the mean ?S.E.of three determinations from a representative of three experiments.

Transcriptional Regulation of SP-A by USF123405

by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

and all contain the sequence encoding the 28amino acids which is lacking in rUSF1b.In the mouse USF1gene sequence,the 84bp of DNA encoding these 28amino acids are located at the 3?-end of exon 6(30).An alternative splice site present within exon 6can be used to splice out the 84nucleotides and create a donor site that can link to the acceptor site at the 5?-end of exon 7without causing a shift in the reading frame.The 84-nucleotide region in rUSF1a cDNA which is deleted in rUSF1b is bracketed by the splice junction consensus nucle-otides AG:GT.

Although USF1is a ubiquitously expressed transcription factor (23),it has been suggested to be involved in the regula-tion of tissue-specific expression of a growing number of genes (31–41).USF1also is suggested to be involved in developmen-tal regulation of Xenopus transcription factor IIIA gene expres-sion (42).By use of RT-PCR,we found the same two species of rUSF1mRNAs in all tissues,corresponding in size to rUSF1a and rUSF1b.In all tissues studied,rUSF1b was the predomi-nant species.Interestingly,rUSF1binding activity was found to be present at high levels in type II epithelial cells but was barely detectable in fibroblasts isolated from fetal lung,sug-gesting a lung cell-specific function of USF1.Considering that USF1s contain two protein interaction domains (helix-loop-helix and leucine zipper),it is possible that USF1s might in-teract with different tissue-specific factors to activate expres-sion of different genes in different tissues.

Transcriptional activity of the SP-A gene is developmentally regulated in fetal rabbit lung and is first detectable in lung nuclei on day 24,reaching maximal levels by day 28of the 31-day gestational period (3).By use of quantitative RT-PCR,we observed that rUSF1a and rUSF1b mRNAs reach peak levels on day 23of gestation just before the time of activation of SP-A gene transcription (3,23).Interestingly,the ratio of rUSF1a to rUSF1b also reaches maximal levels at this time.In previous studies,we observed that binding activity for the DBE and PBE of fetal rabbit lung nuclear proteins also reaches maximal levels on day 24of gestation (5).These findings sug-gest that increased expression of rUSF1s and changes in alter-native splicing which yield a relative increase in rUSF1a may serve an important role in the initiation of SP-A gene expres-sion in fetal rabbit lung during development.Since the 28-amino acid deletion in rUSF1b is located in the second trans-activation domain,it is possible that rUSF1a has increased transactivation potential compared with rUSF1b.Whether a change in the ratio of rUSF1a to rUSF1b serves a role to regulate SP-A gene transcription in fetal lung during develop-ment is uncertain because we observed that cotransfected rUSF1a and rUSF1b have similar efficiencies in transactivat-ing SP-A:hGH fusion genes in A549lung adenocarcinoma cells (Fig.9).

The transactivation domains in USF1have been reported to be relatively weak (40);therefore,heterodimerization of USF1a and USF1b with other transcription factors may be critical in the regulation of SP-A gene expression.Preliminary findings indicate that rUSF1s interact with a related bHLH-LZ protein,USF2,upon binding to the DBE and PBE.2In previous studies,we characterized a CRE-like sequence (6,7)and a GT-box (43)which are also required for basal and cyclic AMP regulation of SP-A promoter activity in transfected type II cells.Our findings suggest that a member of the nuclear receptor family interacts

with the CRE-like sequence (7)and that Sp1and related fac-tors bind to the GT box (43).It,therefore,is likely that regu-lation of SP-A expression in lung type II cells is dependent upon the cooperative interaction of USF1s with the CRE-and GT box-binding proteins.Characterization of the USF1-inter-acting transcription factors and investigation of their regula-tion may provide further insight into the molecular mecha-nisms involved in the developmental,type II cell-specific,and cyclic AMP regulation of SP-A gene expression.

REFERENCES

1.Hawgood,S.,and Schiffer,K.(1991)Annu.Rev.Physiol.53,375–394

2.Auten,R.L.,Watkins,R.H.,Shapiro,D.L.,and Horowitz,S.(1991)Am.J.

Respir.Cell Mol.Biol.3,491–496

3.Boggaram,V.,Qing,K.,and Mendelson C.R.(1988)J.Biol.Chem.263,

2939–2947

4.Mendelson,C.R.,Alcorn,J.L.,and Gao,E.(1993)Semin.Perinatol.17,

223–232

5.Gao, E.,Alcorn,J.L.,and Mendelson, C.R.(1993)J.Biol.Chem.268,

19697–19709

6.Alcorn,J.L.,Gao,E.,Chen,Q.,Smith,M.E.,Gerard,R.D.,and Mendelson,

C.R.(1993)Mol.Endocrinol.7,1072–1085

7.Michael,L. F.,Alcorn,J.L.,Gao, E.,and Mendelson, C.R.(1996)Mol.

Endocrinol.10,8159–8170

8.Singh,H.,Clerc,R.G.,and LeBowitz,J.H.(1989)BioTechniques 7,252–2619.Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989)Molecular Cloning:A

Laboratory Manual ,2nd Ed.,Cold Spring Harbor Laboratory,Cold Spring Harbor,NY

10.Smith,D.B.,and Johnson,K.S.(1988)Gene (Amst.)67,31–40

11.Harlow,E.,and Lane,D.(1988)Antibodies:A Laboratory Manual ,Cold Spring

Harbor Laboratory,Cold Spring Harbor,NY

12.Gorski,K.,Carneiro,M.,and Schibler,U.(1986)Cell 47,767–77613.Meyer,T.E.,and Habener,J.F.(1993)Endocr.Rev.14,269–290

14.Rose,M.D.,Winston,F.,and Hieter,P.(1988)Laboratory Course Manual for

Methods in Yeast Genetics ,Cold Spring Harbor Laboratory,Cold Spring Harbor,NY

15.Lieber,M.,Smith,B.,Szakal,A.,Nelson-Ress,W.,and Todaro,G.(1976)Int.

J.Cancer 17,62–70

https://www.sodocs.net/doc/aa16944793.html,ler,J.H.(1972)Experiments in Molecular Genetics ,Cold Spring Harbor

Laboratory,Cold Spring Harbor,NY

17.Kirschbaum,B.J.,Pognone,P.,and Roeder,R.G.(1992)Mol.Cell.Biol.12,

5094–5101

18.Carthew,R.W.,Chodosh,L.A.,and Sharp,P.A.(1987)Genes Dev.1,937–98019.Sawadogo,M.,and Roeder,R.G.(1985)Cell 43,165–175

20.Bresnick,E.H.,and Felsenfeld,G.(1994)J.Biol.Chem.269,21110–2111621.Ferre-D’Amare,A.R.,Pognonec,P.,Roeder,R.G.,and Burley,S.K.(1994)

EMBO J.13,180–189

22.Bartel,P.L.,Chien,C.-T.,Sternglanz,R.,and Fields,S.(1993)in Cellular

Interactions in Development:A Practical Approach (Harley,D.A.,ed)pp.153–179,Oxford University Press,Oxford

23.Sirito,M.,Lin,Q.,Maity,T.,and Sawadogo,M.(1994)Nucleic Acids Res.22,

427–433

24.Boggaram,V.,and Mendelson,C.R.(1988)J.Biol.Chem.263,19060–1906525.Carthew,R.W.,Chodosh,L.A.,and Sharp,P.A.(1985)Cell 43,439–44826.Gregor,P.D.,Sawadogo,M.,and Roeder,R.G.(1990)Genes Dev.4,1730–174027.Sawadogo,M.(1988)J.Biol.Chem.263,11994–12001

28.Sawadogo,M.,Van Dyke,M.W.,Gregor,P.D.,and Roeder,R.G.(1988)

J.Biol.Chem.263,11985–11993

29.Shieh,B.H.,Sparkes,R.S.,Gaynor,R.B.,and Lusis,A.J.(1993)Genomics 16,

266–268

30.Henrion,A.A.,Vaulont,S.,Raymondjean,M.,and Kahn,A.(1996)Mamm.

Genome 7,803–809

31.Chodosh,L.A.,Carthew,R.W.,Morgan,J.G.,Crabtree,G.R.,and Sharp,

P.A.(1987)Science 238,684–688

32.Bresnick,E.H.,and Felsenfeld,G.(1993)J.Biol.Chem.268,18824–1883433.Reisman,D.,and Rotter,V.(1993)Nucleic Acids Res.21,345–350

34.Outram,S.V.,and Owen,M.J.(1994)J.Biol.Chem.269,26525–26530

35.Cvekl,A.,Sax,C.M.,Bresnick,E.H.,and Piatigorsky,J.(1994)Mol.Cell.Biol.

14,7363–7376

36.Navankasattusas,S.,Sawadogo,M.,van Bilsen,M.,Dang,C.V.,and Chien,

K.R.(1994)Mol.Cell.Biol.14,7331–7339

37.Timchenko,N.,Wilson, D.R.,Taylor,L.R.,Abdelsayed,S.,Wilde,M.,

Sawadogo,M.,and Darlington,G.J.(1995)Mol.Cell.Biol.15,1192–1202

38.Paterson,J.M.,Morrison,C.F.,Mendelson,S.C.,McAllister,J.,and Quinn,

J.P.(1995)Biochem.J.310,401–406

39.Singh,I.S.,Luo,Z.,Kozlowski,M.T.,and Erlichman,J.(1994)Mol.Endocri-nol.8,1163–1174

40.Cogswell,J.P.,Godlevski,M.M.,Bonham,M.,Bisi,J.,and Babiss,L.(1995)

Mol.Cell.Biol.15,2782–2790

41.Carter,R.S.,Ordentlich,P.,and Kadesch,T.(1997)Mol.Cell.Biol.17,18–2342.Kaulen,H.,Pognonec,P.,Gregor,P.D.,and Roeder,R.G.(1991)Mol.Cell.

Biol.11,412–424

43.Young,P.P.,and Mendelson,C.R.(1997)Mol.Endocrinol.,11,1082–1093

2

E.Gao,Y.Wang,and C.R.Mendelson,unpublished observations.

Transcriptional Regulation of SP-A by USF1

23406 by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

Erwei Gao, Ying Wang, Joseph L. Alcorn and Carole R. Mendelson

Expression of the Surfactant Protein-A Gene

The Basic Helix-Loop-Helix-Zipper Transcription Factor USF1 Regulates

doi: 10.1074/jbc.272.37.23398

1997, 272:23398-23406.J. Biol. Chem.

https://www.sodocs.net/doc/aa16944793.html,/content/272/37/23398Access the most updated version of this article at

Alerts:

When a correction for this article is posted ?

When this article is cited ? to choose from all of JBC's e-mail alerts

Click here

https://www.sodocs.net/doc/aa16944793.html,/content/272/37/23398.full.html#ref-list-1This article cites 38 references, 20 of which can be accessed free at by guest on March 2, 2016

https://www.sodocs.net/doc/aa16944793.html,/Downloaded from

机械制造基础形成性考核册作业 答案

机械制造基础形成性考核册作业答案 1、举例说明生产纲领在生产活动中的作用，说明划分生产类型的规律。 答：产品的年生产纲领是指企业在计划期内应当生产的产品产量和进度计划。 在计算出零件的生产纲领以后，即可根据生产纲领的大小，确定相应的生产类型。2、何谓机床夹具?夹具有哪些作用? 答：在机械加工中，为了保证工件加工精度，使之占有确定位置以接受加工或检测的工艺装备统称为机床夹具，简称夹具。 作用：1）保证产品加工精度，稳定产品质量。 2）提高生产效率，降低加工成本。 3）改善工人的劳动条件。 4）扩大机床的工艺范围。 3、机床夹具有哪几个组成部分？各起何作用？ 答：机床夹具大致可以分为6部分。 1）定位部分：用以确定工件在夹具中的正确位置。 2）夹紧元件：用以夹紧工件，确保工件在加工过程中不因外力作用而破坏其定位 精度。 3）导向、对刀元件：用以引导刀具或确定刀具与被加工工件加工表面间正确位 置。 4）连接元件：用以确定并固定夹具本身在机床的工作台或主轴上的位置。 5）夹具体：用以连接或固定夹具上各元件使之成为一个整体。 6）其他装置和元件。 4、工件夹紧的基本要求是什么？ 答：1）夹紧既不应破坏工件的定位，又要有足够的夹紧力，同时又不应产生过大的夹紧变形，不允许产生振动和损伤工件表面。 2）夹紧动作迅速，操作方便、安全省力。 3）手动夹紧机构要有可靠的自锁性；机动夹紧装置要统筹考虑其自锁性和稳定的原动力。 4）结构应尽量简单紧凑，工艺性要好。 5、什么叫“六点定位原则”?什么是欠定位?过定位? 答：夹具用合理分布的六个支承点限制工件的六个自由度，即用一个支承点限制工件的一个自由度的方法，使工件在夹具中的位置完全确定，这就是六点定位原理。 根据工件的加工要求，应该限制的自由度没有完全被限制的定位，称为欠定位。 同一个自由度被几个支承点重复限制的情况，称为过定位（也称为重复定位、超定位）

行政组织学形考任务一参考答案

行政组织学形考任务一 参考答案 标准化管理处编码[BBX968T-XBB8968-NNJ668-MM9N]

行政组织学形考任务一 一、判断题（正确划“√”，错误划“×”，每小题2分，共40分） 1、在霍桑试验的基础上，梅奥于1933年出版了《工业文明中的人的问题》一 书，系统地阐述了与古典管理理论截然不同的一些观点。（√） 2、阿吉里斯在《个性与组织》一书中提出了“不成熟—成熟理论”。（√） 3、斯蒂格利茨由于在决策理论研究方面的贡献而荣获1978年诺贝尔经济学 奖。（×） 4、马斯洛在其代表性着作《人类动机的理论》和《激励与个人》中，提出了着 名的公平理论。（×） 5、美国学者巴纳德在1938年出版的《经理人员的职能》这本书中，系统地提 出了动态平衡组织理论。（√） 6、社会系统组织理论的创始者为美国着名的社会学家罗森茨韦克。（×） 7、邓肯将组织环境分为内部环境和外部环境。（√） 8、卡斯特和罗森茨韦克将影响一切组织的一般环境特征划分为文化特征、技术 特征、教育特征、政治特征、法制特征、自然资源特征、人口特征、社会特征、经济特征等几个方面。（√） 9、组织界限以内与组织的个体决策行为直接相关的自然和社会因素被称为组织

的内部环境。（√） 10、组织界限之外与组织内个体决策直接相关的自然和社会因素被称为组织的外 部环境。（√） 11、“组织”一词，源自希腊文，1873年，哲学家斯宾塞用“组织”来指涉 “已经组合的系统或社会”。（√） 12、与个别行政组织的决策转换过程相关联的更具体的力量被称为行政组织的工 作环境。（√） 13、以明文规定的形式确立下来，成员具有正式分工关系的组织为非正式组织。 （×） 14、以镇压、暴力等控制手段作为控制和管理下属的主要方式，此种类型的组织 为规范性组织。（×） 15、以组织的参与者或成员为主要的受惠对象，组织的目的在于维护及促进组织 成员所追求的利益，此种类型的组织为互利性组织。（√） 16、规范地讲，行政组织是追求行政权力的组织。（×） 17、韦伯是科学管理运动的先驱者，被誉为“科学管理之父”。（×） 18、1911年，泰勒发表了《科学管理原理》一书，掀起了一场科学管理的革 命。（√） 19、行政管理学派的代表人物法约尔，被誉为“管理理论之父”。（√）

机械制造基础形成性考核第四次作业答案

1、举例说明生产纲领在生产活动中的作用，说明划分生产类型的规律。 答：产品的年生产纲领是指企业在计划期内应当生产的产品产量和进度计划。 在计算出零件的生产纲领以后，即可根据生产纲领的大小，确定相应的生产类型。 2、何谓机床夹具?夹具有哪些作用? 答：在机械加工中，为了保证工件加工精度，使之占有确定位置以接受加工或检测的工艺装备统称为机床夹具，简称夹具。 作用：1）保证产品加工精度，稳定产品质量。 2）提高生产效率，降低加工成本。 3）改善工人的劳动条件。 4）扩大机床的工艺范围。 3、机床夹具有哪几个组成部分？各起何作用？ 答：机床夹具大致可以分为6部分。 1）定位部分：用以确定工件在夹具中的正确位置。 2）夹紧元件：用以夹紧工件，确保工件在加工过程中不因外力作用而破 坏其定位精度。 3）导向、对刀元件：用以引导刀具或确定刀具与被加工工件加工表面间 正确位置。 4）连接元件：用以确定并固定夹具本身在机床的工作台或主轴上的位置。 5）夹具体：用以连接或固定夹具上各元件使之成为一个整体。 6）其他装置和元件。 4、工件夹紧的基本要求是什么？ 答：1）夹紧既不应破坏工件的定位，又要有足够的夹紧力，同时又不应产生过大的夹紧变形，不允许产生振动和损伤工件表面。 2）夹紧动作迅速，操作方便、安全省力。 3）手动夹紧机构要有可靠的自锁性；机动夹紧装置要统筹考虑其自锁性和稳定的原动力。 4）结构应尽量简单紧凑，工艺性要好。 5、什么叫“六点定位原则”?什么是欠定位?过定位? 答：夹具用合理分布的六个支承点限制工件的六个自由度，即用一个支承点限制工件的一个自由度的方法，使工件在夹具中的位置完全确定，这就是六点定位原理。 根据工件的加工要求，应该限制的自由度没有完全被限制的定位，称为欠定位。

《行政组织学》形考任务试题及答案学习资料

国开行管专科网络核心课程《行政组织学》形考任务试题及答案 形考任务一 一、判断题（正确划“√”，错误划“×”，每小题2分，共40分） 1.在霍桑试验的基础上，梅奥于1933年出版了《工业文明中的人的问题》一书，系统地阐述了与古典管理理论截然不同的一些观点。\\对 2.阿吉里斯在《个性与组织》一书中提出了“不成熟-—成熟理论”。\\对 3.斯蒂格利茨由于在决策理论研究方面的贡献而荣获1978年诺贝尔经济学奖。\\错 4.马斯洛在其代表性著作《人类动机的理论》和《激励与个人》中，提出了著名的公平理论。\\错 5.美国学者巴纳德在1938年出版的《经理人员的职能》这本书中，系统地提出了动态平衡组织理论\对 6.社会系统组织理论的创始者为美国著名的社会学家罗森茨韦克。\\错 7.邓肯将组织环境分为内部环境和外部环境。\\对 8.卡斯特和罗森茨韦克将影响一切组织的一般环境特征划分为文化特征、技术特征、教育特征、政治特征、法制特征、自然资源特征、人口特征、社会特征、经济特征等几个方面。\\对 9.组织界限以内与组织的个体决策行为直接相关的自然和社会因素被称为组织的内部环境。\\对 10.组织界限之外与组织内个体决策直接相关的自然和社会因素被称为组织的外部环境。\\对 11.“组织”一词，源自希腊文，1873年，哲学家斯宾塞用“组织”来指涉“已经组合的系统或社会”。\\对 12.与个别行政组织的决策转换过程相关联的更具体的力量被称为行政组织的工作环境。\\对 13.以明文规定的形式确立下来，成员具有正式分工关系的组织为非正式组织。\\错 14.以镇压、暴力等控制手段作为控制和管理下属的主要方式，此种类型的组织为规范性组织。\\错 15.以组织的参与者或成员为主要的受惠对象，组织的目的在于维护及促进组织成员所追求的利益，此种类型的组织为互利性组织。\\对 16.规范地讲，行政组织是追求行政权力的组织。\\错 17.韦伯是科学管理运动的先驱者，被誉为“科学管理之父”。\\错 18.1911年，泰勒发表了《科学管理原理》一书，掀起了一场科学管理的革命。\\对 19.行政管理学派的代表人物法约尔，被誉为“管理理论之父”。 \\对 20.德国著名的社会学家韦伯在《高级管理人员的职能》一书中，提出了理想型官僚组织理论。\\错 二、多项选择题（每题有两个和两个以上正确答案，每小题3分，共60分） 21.美国行为科学家赫茨伯格在其《工作的推力》和《工作与人性》等著作中，提出影响人的积极性的因素主要有___。保健因素\\激励因素 22.西蒙指出，决策有两种极端的类型____。程序化决策\\非程序化决策 23.里格斯指出，“棱柱型社会”具有以下三个基本特征____。重叠性\\形式主义\\异质性 24.里格斯在他创立的“棱柱模式理论”中，将社会形态划分____。棱柱社会\\农业社会\\工业社会 25.巴纳德认为，组织不论其级别高低和规模大小，都包含三个基本要素___。共同的目标\\协作的意愿\\信息的联系 26.邓肯将组织环境分为_____。外部环境\\内部环境 27.邓肯从组织环境的___两个维度对影响组织的环境因素进行了深入的分析。静态与动态\\简单与复杂 28.依据学者们的研究，组织的环境分析过程主要包括____等基本阶段。全选 29.伯恩斯和斯塔克将组织结构划分为___。有机式组织结构\\机械式组织结构 30.行政组织环境的基本特点为_____。全选 31.学者们从不同的角度和方法去透视组织，给予不同的定义，目前学界对组织界定的取向，主要有以下几种____。全选 32.依据邓肯的环境模式理论，从简单与复杂、静态与动态两个维度，组织存在的环境状态分别是：___。全选 33.按组织内部是否有正式的分工关系，人们把组织分为____。正式组织\\非正式组织

机械制造基础形考任务一试题及答案整理演示教学

机械制造基础形考任务一试题及答案整理

机械制造基础形考任务一试题及答案整理一、填空题（每空2分，共58分）（请选择正确的文字答案填写，例如：塑性变形） 塑性变形；断裂；变形；破坏；交变载荷；断裂；奥氏体； 渗碳体；断裂前；最大塑性变形；通用橡胶；特种橡胶；含碳量；万分之几；正火；碳钢；合金元素；酸性焊条；碱性焊条；表面淬火； 表面化学热处理；分离工序；整模造型；分模造型；变形工序；挖砂造型；活块造型；焊芯；药皮 题目1. 的能力。 题目2 题目3 性。 题目4 题目5的最大应力值。 题目6优质碳素结构钢的牌号有两位数字表示，这两位数字具体表示钢中 题目7 题目8

题目9 题目10 除，否则会增大钢的淬透性。 题目11 题目12 题目13 题目14 二、是非判断题（每题1分，共42分） 题目15冲击韧性值随温度的降低而增加。 选择一项： 对 错 题目16抗拉强度是表示金属材料抵抗最大均匀塑性变形或断裂的能力。选择一项： 对 错 题目17硬度是指金属材料抵抗其他物体压入其表面的能力。 选择一项： 对 错

题目18金属材料在外载荷作用下产生断裂前所能承受最大塑性变形的能力称为塑性。 选择一项： 对 错 题目19冲击韧性值随温度的降低而减小。 选择一项： 对 错 题目20强度越高，塑性变形抗力越大，硬度值也越高。 选择一项： 对 错 题目21屈服强度是表示金属材料抵抗微量弹性变形的能力。 选择一项： 对 错 题目22冲击韧性值愈大，材料的韧性愈好。 选择一项： 对 错 题目23硬度是指金属材料抵抗比它更硬的物体压入其表面的能力。

选择一项： 对 错 题目24通常材料的力学性能是选材的主要指标。 选择一项： 对 错 题目25一般来说，材料的硬度越高，耐磨性越好。 选择一项： 对 错 题目26测量布氏硬度时，压头为淬火钢球，用符号HBW表示。选择一项： 对 错 题目27测量布氏硬度时，压头为淬火钢球，用符号HBS表示。 选择一项： 对 错 题目28测量布氏硬度时，压头为硬质合金球，用符号HBW表示。选择一项： 对

GENESIS2000入门教程中英文转换

?GENESIS2000入门教程 Padup谷大pad paddn缩小pad reroute 扰线路Shave削pad linedown缩线line/signal线Layer 层in 里面 out外面Same layer 同一层spacing 间隙cu铜皮 Other layer另一层positive 正 negative负Temp 临时 top顶层bot底层Soldermask 绿油层silk字符层 power 电源导(负片) Vcc 电源层(负片) ground 地层(负片) apply 应用 solder 焊锡singnal 线路信号层soldnmask绿油层input 导入 component 元器件Close 关闭zoom放大缩小create 创建 Reste 重新设置corner 直角step PCB 文档Center 中心 snap 捕捉board 板Route 锣 带repair 修理、编辑 resize (编辑)放大缩小analysis 分析Sinde 边、面Advanced 高级 measuer 测量PTH hole 沉铜孔NPTH hole 非沉铜孔output 导出 VIA hole 导通孔smd pad 贴片PAD replace 替换fill 填充 Attribute 属性round 圆square 正方形rectangle 矩形

Select 选择include 包含exclude 不包 含step 工作单元 Reshape 改变形状profile 轮廓drill 钻 带rout 锣带 Actions 操作流程analyis 分析DFM 自动修改编辑circuit 线性 Identify 识别translate 转换job matrix 工作 室repair 修补、改正 Misc 辅助层dutum point 相对原点corner 直 角optimization 优化 origin 零点center 中心global 全 部check 检查 reference layer 参考层reference selection 参考选 择reverse selection 反选 snap 对齐invert 正负调换symbol 元 素feature 半径 histogram 元素exist 存在angle 角 度dimensions 标准尺寸 panelization 拼图fill parameters 填充参 数redundancy 沉余、清除 层英文简写层属性 顶层文字Top silk screen CM1( gtl ) silk-scren 顶层阻焊Top solder mask SM1 ( gts ) solder-mask 顶层线路Top layer L1 ( gtl ) signal 内层第一层power ground (gnd) PG2 ( l2-pw ) power-ground(负片) 内层第二层signal layer L3 signal (正片) 内层第三层signal layer L4 signal (正片)

2018行政组织学形考任务试题

行政组织学形考任务试题 ?第三章行政组织的环境与管理/ ?? ?形考任务1 信息文本 一、判断题（正确划“√”，错误划“×”，每小题2分，共40分） 题目1 题干 在霍桑试验的基础上，梅奥于1933年出版了《工业文明中的人的问题》一书，系统地阐述了与古典管理理论截然不同的一些观点。（） 选择一项： 对 错 题目2 题干 阿吉里斯在《个性与组织》一书中提出了“不成熟-—成熟理论”。（） 选择一项： 对 错

题目3 题干 斯蒂格利茨由于在决策理论研究方面的贡献而荣获1978年诺贝尔经济学奖。（） 选择一项： 对 错 题目4 题干 马斯洛在其代表性著作《人类动机的理论》和《激励与个人》中，提出了著名的公平理论。（） 选择一项： 对 错 题目5 题干 美国学者巴纳德在1938年出版的《经理人员的职能》这本书中，系统地提出了动态平衡组织理论。（）

选择一项： 对 错 题目6 题干 社会系统组织理论的创始者为美国著名的社会学家罗森茨韦克。（）选择一项： 对 错 题目7 题干 邓肯将组织环境分为部环境和外部环境。（） 选择一项： 对 错 题目8

题干 卡斯特和罗森茨韦克将影响一切组织的一般环境特征划分为文化特征、技术特征、教育特征、政治特征、法制特征、自然资源特征、人口特征、社会特征、经济特征等几个方面。（）选择一项： 对 错 题目9 题干 组织界限以与组织的个体决策行为直接相关的自然和社会因素被称为组织的部环境。（）选择一项： 对 错 题目10 题干 组织界限之外与组织个体决策直接相关的自然和社会因素被称为组织的外部环境。（） 选择一项： 对 错

机械制造基础形考任务一试题及答案整理

机械制造基础形考任务一试题及答案整理 一、填空题(每空2分,共58分)(请选择正确的文字答案填写,例如:塑性变形) 塑性变形;断裂;变形;破坏;交变载荷;断裂;奥氏体; 渗碳体;断裂前;最大塑性变形;通用橡胶;特种橡胶;含碳量;万分之几;正火;碳钢;合金元素;酸性焊条;碱性焊条;表面淬火; 表面化学热处理;分离工序;整模造型;分模造型;变形工序;挖砂造型;活块造型;焊芯;药皮 题目1、 题目2强度就是指金属材料在外载荷作用下, 题目3 题目4在铁碳合金中, 题目5的最大应力值。 题目6优质碳素结构钢的牌号有两位数字表示, 题目7 题目8 题目9 题目10淬火前,若钢中存在网状渗碳体,,否则会增大钢的淬透性。 题目11 题目12根据药皮所含氧化物的性质, 题目13 题目14 二、就是非判断题(每题1分,共42分) 题目15冲击韧性值随温度的降低而增加。 选择一项:

对 错 题目16抗拉强度就是表示金属材料抵抗最大均匀塑性变形或断裂的能力。 选择一项: 对 错 题目17硬度就是指金属材料抵抗其她物体压入其表面的能力。 选择一项: 对 错 题目18金属材料在外载荷作用下产生断裂前所能承受最大塑性变形的能力称为塑性。选择一项: 对 错 题目19冲击韧性值随温度的降低而减小。 选择一项: 对 错 题目20强度越高,塑性变形抗力越大,硬度值也越高。 选择一项: 对 错 题目21屈服强度就是表示金属材料抵抗微量弹性变形的能力。 选择一项: 对 错 题目22冲击韧性值愈大,材料的韧性愈好。 选择一项: 对

错 题目23硬度就是指金属材料抵抗比它更硬的物体压入其表面的能力。选择一项: 对 错 题目24通常材料的力学性能就是选材的主要指标。 选择一项: 对 错 题目25一般来说,材料的硬度越高,耐磨性越好。 选择一项: 对 错 题目26测量布氏硬度时,压头为淬火钢球,用符号HBW表示。 选择一项: 对 错 题目27测量布氏硬度时,压头为淬火钢球,用符号HBS表示。 选择一项: 对 错 题目28测量布氏硬度时,压头为硬质合金球,用符号HBW表示。 选择一项: 对 错 题目29测量洛氏硬度时,压头为120°金刚石圆锥体,用符号HRC表示。选择一项: 对 错

GENESIS基础——步骤

新建料号： 导入资料、查看并更正错误: 首先查看层，若出现细线或出现大块的图案为D码有问题！必须在Rep层中点击右键选择D码学习器去修改，打开后出现Wheel Template Editor窗口！若确认是单位错了，就在菜单Parms中选择Global 中修改单位，点击后出现Global Parameters Popup对话框，改了单位后点击Ok即可，然后Actions 菜单中选择Translate Wheel执行D码文件，若有红色问题，则要手工修改，选中问题点击Sym：，确认形状，输入对应的参数，点击Ok即可，完成此动作，在File中选Closs关闭文件。 用同样的方法一层一层的修改其它问题层，改完后最后修改drl钻带文件。首先确认尺寸，然后在Rep 层右键打开D码学习器去修改，确认单位，若有问题则同上方法修改，然后再查看有否连孔，若有则是格式不对，再查看孔位是否很散，若有则是省零格式错误。常用的几种格式：英制inch、mil有：2:3 2:4 2:.5 3:5公制mm有：3:3 4:4 在钻带层（drl）点击右键选择Aview Ascii查看文字档，看最长的坐标，数X、Y有几位数，看坐标如有八位数则用3.5和4.4去修改，在钻带层点击Parameters中选Numberef Fromat修改小数格式，坐标单位同时跟小数格式一改，同时钻带单位也要和坐标单位一致！ 省零格式：Leading 前省零，None不省零，Trailing 后省零。 Gerber格式通常是前省零，钻带格式通常是后省零。 层命名、排序、定属性： 改完后点击Ok即可，所有格式改完后，打开所有层，执行进去。执行后，打开 Job Matrix特性表命名层名 层对齐： 打开所有影响层，在层名点击右键，选Register对齐，点击后出现Register Layer Popup窗口。在Referenee Layer：中选择参考层线路层。除了文字层和分孔层不能自动对齐外，其它层可自动对齐，自动对齐后马上关闭影响层。单一打开没有对齐的那层，抓中心，出现Sanp Popup窗口，选Center，然后选Edit→Move→Same Layer 同层移动，点OK，再点击外形框左下角，点击右键，接着打开参考层，按S+A 转换工作层，再点击原参考层外型框即可。图形相隔太远的，可以用Ctrl+A暂停，然后框选放大，确定目标时按S+A转换工作层，再电击原参考层左下角即可。 建外形框： 所有层对齐后，打开分孔图，用网选命令选中外型框，用Edit→Copy→Other Layer 复制到新层，重新命名层名为gko（外型框），点击OK。单一打开gko，框选板内所有不要的东西删除，改单位，然后用Edit→Reshape→Change Symbol更改符号，出现Chang Feetar窗口，其中Symbol（外型线线粗）：R200。建Profile虚线： 更改后，用网选命令选中外型框，用Edit→Create→Profile创建虚线。

国开《行政组织学》形考任务试题及答案

国开《行政组织学》形考任务试题及答案 形考任务一 一、判断题 1.在霍桑试验的基础上，梅奥于1933年出版了《工业文明中的人的问题》一书，系统地阐述了与古典管理理论截然不同的一些观点。\\对 2.阿吉里斯在《个性与组织》一书中提出了“不成熟-—成熟理论”。\\对 3.斯蒂格利茨由于在决策理论研究方面的贡献而荣获1978年诺贝尔经济学奖。\\错 4.马斯洛在其代表性著作《人类动机的理论》和《激励与个人》中，提出了著名的公平理论。\\错 5.美国学者巴纳德在1938年出版的《经理人员的职能》这本书中，系统地提出了动态平衡组织理论\对 6.社会系统组织理论的创始者为美国著名的社会学家罗森茨韦克。\\错 7.邓肯将组织环境分为内部环境和外部环境。\\对 8.卡斯特和罗森茨韦克将影响一切组织的一般环境特征划分为文化特征、技术特征、教育特征、政治特 征、法制特征、自然资源特征、人口特征、社会特征、经济特征等几个方面。\\对 9.组织界限以内与组织的个体决策行为直接相关的自然和社会因素被称为组织的内部环境。\\对 10.组织界限之外与组织内个体决策直接相关的自然和社会因素被称为组织的外部环境。\\对 11.“组织”一词，源自希腊文，1873年，哲学家斯宾塞用“组织”来指涉“已经组合的系统或社会”。\\对 12.与个别行政组织的决策转换过程相关联的更具体的力量被称为行政组织的工作环境。\\对 13.以明文规定的形式确立下来，成员具有正式分工关系的组织为非正式组织。\\错 14.以镇压、暴力等控制手段作为控制和管理下属的主要方式，此种类型的组织为规范性组织。\\错 15.以组织的参与者或成员为主要的受惠对象，组织的目的在于维护及促进组织成员所追求的利益，此种类型的组织为互利性组织。\\对 16.规范地讲，行政组织是追求行政权力的组织。\\错 17.韦伯是科学管理运动的先驱者，被誉为“科学管理之父”。\\错 18.1911年，泰勒发表了《科学管理原理》一书，掀起了一场科学管理的革命。\\对 19.行政管理学派的代表人物法约尔，被誉为“管理理论之父”。 \\对 20.德国著名的社会学家韦伯在《高级管理人员的职能》一书中，提出了理想型官僚组织理论。\\错

GENESIS 菜单入门教程

GENESIS2000入门教程 Padup谷大pad paddn缩小pad reroute 扰线路Shave削pad linedown缩线line/signal线Layer 层 in 里面 out外面Same layer 同一层spacing 间隙 cu 铜皮 Other layer另一层positive 正negative负 Temp 临时 top 顶层bot 底层Soldermask 绿油层 silk 字符层 power 电源导(负片) Vcc 电源层(负片) ground 地层(负片) apply 应用 solder 焊锡singnal 线路信号层 soldnmask绿油层 input 导入 component 元器件Close 关闭zoom放大缩小create 创建 Reste 重新设置corner 直角step PCB文档

Center 中心 snap 捕捉board 板Route 锣带repair 修理、编辑 resize (编辑)放大缩小analysis 分析Sinde 边、面Advanced 高级 measuer 测量PTH hole 沉铜孔 NPTH hole 非沉铜孔output 导出 VIA hole 导通孔smd pad 贴片PAD replace 替换fill 填充 Attribute 属性round 圆square 正方形rectangle 矩形 Select 选择include 包含exclude 不包含step 工作单元 Reshape 改变形状profile 轮廓drill 钻带rout 锣带 Actions 操作流程 analyis 分析 DFM 自动修改编辑circuit 线性 Identify 识别 translate 转换 job matrix 工作室

行政组织学形考任务5

形考任务五 一、判断题（正确划“√”，错误划“×”，每小题2分，共40分） 1.根据行政组织文化产生的时间，行政组织文化可以分为传统行政组织文化和当代行政组织文化。对 2.行政组织文化具有多种功能，它能把组织成员个人目标与组织目标有机结合起来，引导组织成员的行为，我们把这种功能称为控制功能。错 3.行政组织文化相比于正式的组织规章制度的控制作用，它具有软约束性的特性。对 4.行政组织文化是一种群体文化，是一种无形的管理方式。对 5.行政组织绩效就是指的行政组织活动的成果。错 6.经济性指标一般指行政组织投入到管理中的资源，其关心的是行政组织的投入。对 7.效果通常是指公共服务符合政策目标的程度，其关心的是手段。对 8.效率就是指投入与产出之间的比例，力求以最少的投入获得最大的产出，其关心的是手段问题。对 9.组织变革不是一个持续循环与发展的过程，因为要考虑到组织的稳定。对 10.组织发展起源于20世纪50年代初的调查反馈方法和实验室培训运动。它的先驱是法国心理学家烈文。对 11.1957年麦格雷戈应邀到联合碳化公司与公司人事部门联合成立顾问小组，把实验室训练的技术系统地在公司使用。这个小组后被称之

为“T训练小组”。错 12.作为一套极有系统的组织发展方案，格道式发展模式的目的在于使组织达到一种最佳状态。此模式创立者为布莱克和默顿。对 13.系统变革模式认为，组织是一个系统，是由技术、结构、人员和任务四个因素构成，任何一个因素的变化都会牵动和引起系统的变化。系统变革模式的创始人为利维特。对 14.美国心理学家埃德加·薛恩在其《组织心理学》一书中提出了系统变革模式。错 15.罗宾·斯特克兹认为，组织变革的方式取决于组织成员的技术能力和人际关系能力的组合，提出了渐进式变革模式。错 16.管理学大师德鲁克在《后资本主义社会》一书中指出：“世界上没有贫穷的国家，只有无知的国家”。对 17.知识经济与传统经济相比，知识成为组织根本的生产要素。对 18.组织理论家卡斯特和罗森茨韦克认为，未来的组织将更趋向于动态和灵活。对 19.战略管理的核心是问题管理。错 20.随着信息技术的发展，将信息科技运用于行政组织的管理，建立“节约型政府”已经成为各国的一个普遍趋势。错 二、多项选择题（每题有两个和两个以上正确答案，每小题3分，共60分） 21.根据其在行政组织中所占有的地位，行政组织文化可以分为___。主文化亚文化

江苏开放大学机械制造基础形考1

江苏开放大学 形成性考核作业学号 姓名 课程代码110036 课程名称机械制造基础评阅教师 第 1 次任务 共 5 次任务 江苏开放大学

一、选择题 1、金属材料在外力作用下抵抗塑性变形和断裂的能力叫（ B ）。 A．硬度 B．强度 C．塑性 D．弹性 2、高碳钢获得最佳切削性能的热处理工艺方法是（ B ）。 A.完全退火 B.球化退火 C.去应力退火 D.正火 3、08F钢中的平均含碳量为（ A ）。 A．0.08% B.0.8% C.8% 4、T10A钢按用途分属于（ C ）。 A．沸腾钢 B.优质钢 C.工具钢 5、在常用材料中，灰铸铁铸造性能（ A ）。 A．好 B．差 C．较差 6、无需填充金属，焊接变形小，生产率高的焊接方法是（ B ）。 A．埋弧焊 B．电阻焊 C．电弧焊 7、下列材料中（ B ）材料的焊接性能最差。 A．低碳钢 B．高碳钢 C．铸铁 8、机床床身的材料一般为（ B ）。 A．可锻铸铁 B.灰铸铁 C.球墨铸铁 D.蠕墨铸铁 9、下列金属属于轴承合金的是（ A ）。 A．铅青铜 B.硅青铜 C.铝镁合金 D.铝锌合金 10、生产柴油机曲轴应选材料是（ C ）。 A．HT200 B.QT700-2 C.KTH330-08 D.Q345 二、填空题 1、纯铁在加热过程中，其体心立方晶格与面心立方晶格的相互转变现象，称为同素异构转变。 2、一般情况下，晶粒越细，金属的强度、塑性和韧性越好。 3、奥氏体是碳在γ-Fe中的固溶体，呈面心立方晶格。 4、根据Fe-Fe3C 相图中成分-组织-性能的规律，选材时，建筑结构和各种型钢选塑性和韧性好的低碳钢，各种机械零件选用强度、塑性和韧性好的中碳钢，各种工具要用硬度高而耐磨性好的高碳钢。

《行政组织学》形考任务一答案

《行政组织学》形考任务一 一、判断题（正确划“√”，错误划“×”，每小题2分，共40分） 在霍桑试验的基础上，梅奥于1933年出版了《工业文明中的人的问题》一书，系统地阐述了与古典管理理论截然不同的一些观点。（√） 2. 阿吉里斯在《个性与组织》一书中提出了“不成熟-—成熟理论”。（√） 3.斯蒂格利茨由于在决策理论研究方面的贡献而荣获1978年诺贝尔经济学奖。（× ） 4.马斯洛在其代表性著作《人类动机的理论》和《激励与个人》中，提出了著名的公平理论。（× ） 5.美国学者巴纳德在1938年出版的《经理人员的职能》这本书中，系统地提出了动态平衡组织理论。（√） 6.社会系统组织理论的创始者为美国著名的社会学家罗森茨韦克。（× ） 7.邓肯将组织环境分为内部环境和外部环境。（√） 8.卡斯特和罗森茨韦克将影响一切组织的一般环境特征划分为文化特征、技术特征、教育特征、政治特征、法制特征、自然资源特征、人口特征、社会特征、经济特征等几个方面。（√） 9.组织界限以内与组织的个体决策行为直接相关的自然和社会因素被称为组织的内部环境。（√） 10.组织界限之外与组织内个体决策直接相关的自然和社会因素被称为组织的外部环境。（√） 11.“组织”一词，源自希腊文，1873年，哲学家斯宾塞用“组织”来指涉“已经组合的系统或社会”。（√）

12.与个别行政组织的决策转换过程相关联的更具体的力量被称为行政组织的工作环境。（√） 13.以明文规定的形式确立下来，成员具有正式分工关系的组织为非正式组织。（× ） 14.以镇压、暴力等控制手段作为控制和管理下属的主要方式，此种类型的组织为规范性组织。（× ） 15.以组织的参与者或成员为主要的受惠对象，组织的目的在于维护及促进组织成员所追求的利益，此种类型的组织为互利性组织。（√ ） 16.规范地讲，行政组织是追求行政权力的组织。（× ） 17.韦伯是科学管理运动的先驱者，被誉为“科学管理之父”。（× ） 18.1911年，泰勒发表了《科学管理原理》一书，掀起了一场科学管理的革命。（√ ） 19.行政管理学派的代表人物法约尔，被誉为“管理理论之父”。（√） 20.德国著名的社会学家韦伯在《高级管理人员的职能》一书中，提出了理想型官僚组织理论。（× ） 二、多项选择题（每题有两个和两个以上正确答案，每小题3分，共60分） 21.美国行为科学家赫茨伯格在其《工作的推力》和《工作与人性》等著作中，提出影响人的积极性的因素主要有______AC______。 A. 激励因素 B. 物质因素 C. 保健因素 D. 精神因素

GENESIS脚本编程教材PERL

Perl学习笔记 (2) 1.Perl简介 (2) 2.数据类型 (4) 2.1概览 (4) 2.2命名空间(Namespaces)4 2.3标量(Scalars)5 2.4数组(Arrays)8 2.5关联数组(Hashes)11 2.6引用(References)12 2.6.1Perl引用简介 (12) 2.6.2创建引用 (12) 2.6.3使用引用 (13) 2.6.4符号引用 (14) 2.6.5垃圾回收与弱引用 (15) 2.7数据结构 (16) 2.7.1Arrays of Arrays16 2.7.2Hash of Arrays18 2.7.3Arrays of Hashes20 2.7.4Hashes of Hashes22 2.7.5Hashes of Functions24 3操作符（Operators） (25) 3.1概述 (25)

3.2Perl操作符一览 (25) 3.3各种操作符使用说明 (27) 3.3.1项与左赋列表操作符 (27) 3.3.2箭头操作符 (27) 3.3.3自增自减 (27) 3.3.4乘方 (27) 3.3.5表意一元操作符 (28) 3.3.6捆绑操作符 (28) 3.3.7乘操作符 (28) 3.3.8加操作符 (28) 3.3.9移位操作符 (29) 3.3.9有名一元和文件测试操作符 (29) 3.3.10关系操作符 (30) 3.3.11位操作符 (31) 3.3.12C风格逻辑操作符 (31) 3.3.13范围操作符 (31) 3.3.14条件操作符 (31) 3.3.14赋值操作符 (32) 3.3.15逗号操作符 (32) 3.3.16逻辑and,or,not和xor操作符 (33) 3.4与C操作符的比较 (33) 3.4.1Perl操作符的特别之处 (33)

国家开放大学《行政组织学》形考任务1试题

国家开放大学《行政组织学》形考任务1试题 在霍桑试验的基础上，梅奥于1933年出版了《工业文明中的人的问题》一书，系统地阐述了与古典管理理论截然不同的一些观点。（） 阿吉里斯在《个性与组织》一书中提出了不成熟 斯蒂格利茨由于在决策理论研究方面的贡献而荣获1978年诺贝尔经济学奖。（） 马斯洛在其代表性著作《人类动机的理论》和《激励与个人》中，提出了著名的公平理论。 美国学者巴纳德在1938年出版的《经理人员的职能》这本书中，系统地提出了动态平衡组织理论。（） 社会系统组织理论的创始者为美国著名的社会学家罗森茨韦克。（） 邓肯将组织环境分为内部环境和外部环境。（） 卡斯特和罗森茨韦克将影响一切组织的一般环境特征划分为文化特征、技术特征、教育特征、政治特征、法制特征、自然资源特征、人口特征、社会特征、经济特征等几个方面。（） 组织界限以内与组织的个体决策行为直接相关的自然和社会因素被称为组织的内部环境。 组织界限之外与组织内个体决策直接相关的自然和社会因素被称为组织的外部环境。（） 组织一词，源自希腊文，1873年，哲学家斯宾塞用组织来指涉已经组合的系统或社会。（） 与个别行政组织的决策转换过程相关联的更具体的力量被称为行政组织的工作环境。（） 以明文规定的形式确立下来，成员具有正式分工关系的组织为非正式组织。（） 以镇压、暴力等控制手段作为控制和管理下属的主要方式，此种类型的组织为规范性组织。 以组织的参与者或成员为主要的受惠对象，组织的目的在于维护及促进组织成员所追求的利益，此种类型的组织为互利性组织。（） 规范地讲，行政组织是追求行政权力的组织。（） 韦伯是科学管理运动的先驱者，被誉为科学管理之父。（） 1911年，泰勒发表了《科学管理原理》一书，掀起了一场科学管理的革命。（） 行政管理学派的代表人物法约尔，被誉为管理理论之父。（） 德国著名的社会学家韦伯在《高级管理人员的职能》一书中，提出了理想型官僚组织理论。 美国行为科学家赫茨伯格在其《工作的推力》和《工作与人性》等著作中，提出影响人的

机械制造基础形成性考核册作业1答案

机械制造基础形成性考核册作业1答案 1、常用的工程材料可以用教材第一页的表格表示，请完成工程材料的分类表： 答： 1、人们在描述金属材料力学性能重要指标时，经常使用如下术语，请填写其使用的符号和 内涵： （a）强度：金属材料在外载荷的作用下抵抗塑性变形和断裂的能力。强度有屈服强度σs和抗拉强度σb。 （b）塑性：金属材料在外载荷作用下产生断裂前所能承受最大塑性变形的能力。 （c）强度：是指金属材料抵抗比它更硬的物体压入其表面的能力。硬度有布氏硬度HBS和洛氏硬度HR。 （d）冲击韧性：金属抵抗冲击载荷作用而不被破坏的能力。 （e）疲劳强度：金属材料经受无数次交变载荷作用而不引起断裂的最大应力值。2、参照教材图1—2填写材料拉伸曲线中的相应特征值点的符号，并描述相应的含义。 （a）横坐标表示：试件的变形量ΔL （b）纵坐标表示：载荷F （c）S点：屈服点 （d）b点：颈缩点 （e）k点：拉断点 （f）屈服点以后出现的规律：试样的伸长率又随载荷的增加而增大，此时试样已产生较大的塑性变形，材料的抗拉强度明显增加。 3、一根标准试样的直径为10mm、标距长度为50mm。拉伸试验时测出试样在26kN时屈服， 出现的最大载荷为45 kN。拉断后的标距长度为58mm，断口处直径为7.75mm。试计算试样的σ0.2、σb。 答：σ0.2=F0.2/S0=26000/(3.14*0.0052)=3.3*108MPa σb=F b/S0=45000/(3.14*0.0052)=5.7*108MPa

4、HR是零件设计中常用的表示材料硬度指标。请回答下表中表示的有效范围和应用范围： HR和HB有什么差别？ 答：两种都是测试硬度的标准，区别在于测量方法不同。两种硬度标准根本性区别在于：布氏和洛氏测量的对象不同。布氏测量低硬度的材料，洛氏测量高硬度的材料。 6、参照教材1-7图补充如下Fe-Fe3C相图缺少的内容（标注相应的数据和材料相符号），思考每个线条和线条包容区域内金属相符号的特点 参考上图回答以下问题：： （a.）. AC线为：合金的液相线 （b.）. Acm线为：碳在奥氏体中的溶解限度线 （c.）. AC3线为：奥氏体中开始析出铁素体或铁素体全部溶入奥氏体的转变线 （d.）. A表示：奥氏体相 （e.）. F表示：铁素体相 （f.）. P表示：珠光体相 （g.）. Ld表示：液相 （h.）. Fe3C表示：渗碳体相 （i.）. 含碳0.77％为：都是奥氏体 （j.）. 导致过共析钢中材料脆性很高的主要原因是：若加热到略高于AC1温度时，珠光体完全转变成奥氏体，并又少量的渗碳体溶入奥氏体。此时奥氏体晶粒 细小，且其碳的质量分数已稍高与共析成分。如果继续升高温度，则二次渗

genesis 全套最快速制作 操作步骤

Designer By:Anjie Date:2015-09-09 资料整理 1.检查整理资料(解压缩.zip,打印客户PDF等资料). 2.INPUT资料（注意钻孔D-CODE属性设置） 3.更改层命名，定义层属性及排序. 4.层对齐及归原点（最左下角）. 5.存ORG. 整理原始网络 6.钻孔核对分孔图(MAP) 7.挑选成型线至outline层 8.工作层outline层移到0层. 9.整理钻孔(例如：将大于6.4mm钻孔移动到outline层, 其它层NPTH,SLOT移 动到DRL层) 10.整理成型线（断线、缺口、R8） 11.整理outline(将outline层需要钻孔的移动到drl层) 12.创建Profile. 13.板外物移动到0层. 14.核对0层成型线及板外物是否移除正确. 15.内层网络检查（如负性假性隔离） 16.防焊转PAD 17.线路转PAD

18.分析钻孔（检查线路PAD是否有漏孔、重孔修正，内层short） 19.定义SMD属性 20.存NET 21.打印原稿图纸. 编辑钻孔 22.补偿钻孔 （1）检查原始孔径是否正确（不能有“？”号） （2）合刀排序 （3）输入板厚与补偿值（PTH+4 /PTH+6） （4）定义钻孔属性（VIA，PTH，NPTH）主要定义VIA属性NPTH在整理原始网络前定义. （5）输入公差(注意单位). （6）检查最大与最小孔是否符合规范 （7）短SLOT孔分刀，8字孔分刀。（尾数+1 或-1） 23．校对钻孔中心（参照TOP防焊及TOP线路） 24．分析钻孔 25．短SLOT孔加预钻孔 26．挑选NPTH属性的孔移动到新建NPTH层. 内层负片编辑 1．检查有无负性物件（负性物件需要合并） 2．层属性是否为NEG 3．对齐钻孔(内层负片为影响层，参考钻孔层对齐)

行政组织学形考任务四参考答案

一、判断题（正确划“√”，错误划“×”，每小题2分，共40分） 1、行政组织决策的目的是为了实现社会的共同利益。（√） 2、行政组织决策是以行政权力为后盾。√ 3、风险型决策的决策后果无法预测。× 4、确定目标是行政组织进行决策的起点。× 5、中枢系统是行政组织决策的中心。√ 6、美国政治学家伊斯顿被认为是决策理论研究的开创者。× 7、在决策理论研究领域，杜鲁门提出了团体决策模型。√ 8、现代观点认为，冲突既具有建设性又具有破坏性。√ 9、组织中最佳的冲突状态是没有冲突。× 10、解决冲突的基本策略中具有“治本”性的是正视策略。√ 11、合作意向都很高，宁可牺牲自身利益而使对方达到目的的冲突处理模式为协作型。× 12、通过组织明文规定的原则、渠道进行的信息传递和交流，是一种正式沟通。√ 13、信息的发讯者和受讯者以协商、会谈、讨论的方式进行信息的交流与意见反馈，直到双方共同了解为止，这种沟通形式为双向沟通。√ 14、组织系统中处于相同层次的人、群体、职能部门之间进行的信息传递和交流为平行沟通 √ 15、在组织管理中，书面沟通方式要优于口头沟通。× 16、作报告、发指示、下命令等属于单向沟通。√ 17、20世纪90年代初陈国权开始研究组织学习和学习型组织，并提出了组织学习系统理论（OLST）。√ 18、行政组织学习是一种全员学习。× 19、知识的主要构成要素包括经验、事实、判断以及经验法则。√ 20、行政组织学习不是组织内部成员个人学习的简单相加，而是一个社会过程。√ 二、多项选择题（每题有两个和两个以上正确答案，每小题3分，共60分） 21、根据决策所具有的条件的可靠程度的不同，决策可分为____。 确定型决策, 风险型决策, 不确定型决策 22、正确的决策目标应该具备的条件是___。定量化, 有一定的时间限制, 要明确责任 23、西蒙的决策过程包括___。 情报活动阶段, 设计活动阶段, 抉择活动阶段, 审查活动阶段 24、冲突的特性有_____。客观性, 主观性, 程度性 25、符合现代冲突观点的是____。 冲突本身没有好坏之分, 有些冲突对组织具有破坏性, 有些冲突对组织具有建设性 26、根据冲突发生的方向，可将冲突分为_________。 横向冲突, 纵向冲突, 直线/职能冲突 27、回避策略中，解决冲突的方法包括______. 忽视, 分离, 限制 28、减少冲突的策略主要有______。谈判, 设置超级目标, 第三方介入, 结构调整 29、从组织沟通的一般模式和组成要素来看，组织沟通具有以下几个特点：_____。 动态性, 互动性, 不可逆性, 环境制约性 30、以组织结构及其运行程序为依据和标准，组织信息沟通的形式和类型可划分为以下几种：______。下行沟通, 上行沟通, 平行沟通 31、根据沟通是否需要第三者中介传递，我们可将沟通划分为以下两种类型：_____。 直接沟通, 间接沟通