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At least 1,207 records · Page 67Linked to original sources
PubMed1994

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice.

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Decidual/trophoblast prolactin-related protein: characterization of gene structure and cell-specific expression.

Decidual/trophoblast PRL-related protein (d/tPRP) is a member of the PRL gene family and is dually expressed in uterine and placental tissues in a highly coordinated pattern during pregnancy. In the present study, we describe the isolation and characterization of the d/tPRP gene. A lambda DASH II Wistar-Kyoto rat genomic library was screened with a labeled d/tPRP complementary DNA, resulting in the isolation of two phage clones, RGLd-41 [17.7 kilobases (kb)] and RGLd-42 (15.8 kb). RGLd-41 alone was found to contain the full-length d/tPRP gene and was used for subsequent analyses. The d/tPRP gene possesses a six-exon, five-intron organization. Relative to other highly conserved members of the PRL gene family, d/tPRP contains a single small additional exon (exon 3) situated between exons 2 and 3 of the prototypical PRL gene. The region corresponding to exon 3 of d/tPRP encodes for a unique amino acid region found in a subset of PRL family members. A reverse transcription-PCR (RT-PCR) tissue survey for d/tPRP messenger RNA revealed that d/tPRP expression was restricted to decidual and trophoblast tissues. A single transcription start site 65 bp upstream of the initiation codon was identified in decidual tissue, whereas multiple transcription start sites ranging from 61-66 bp upstream of the initiation codon were detected in placental tissue. Various tissue culture systems (primary cultures and cell lines) were evaluated for d/tPRP expression and activation of a 3.96-kb d/tPRP promoter-luciferase reporter construct. Decidual, spongiotrophoblast, and trophoblast giant cell populations expressed d/tPRP and were capable of activating the d/tPRP promoter-reporter construct, whereas other cell types were ineffective. Limited d/tPRP promoter activation was noted in uterine stromal cell lines. In summary, d/tPRP possesses a unique six-exon, five-intron gene structure and exhibits cell-specific expression that is regulated at least in part by a 3.96-kb 5'-flanking region.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the gene for the b subunit of human factor XIII.

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.

Amino Acid Sequence↗
PubMed1996

Identification of Pasteurella multocida tryptophan synthase beta-subunit by antisera against strain P1059.

Pasteurella multocida strain P1059 is a highly virulent bacterium which causes fowl cholera in turkeys and chickens. A genomic library of P. multocida P1059 DNA was constructed using pUC19, expressed in Escherichia coli DH5 alpha, and screened with chicken antisera generated against P. multocida P1059. Twelve out of the 4100 clones screened were immunoreactive. Plasmids isolated from these twelve clones were transformed into E. coli CSR603 for maxicell analysis. Five proteins, with molecular masses of 34, 37, 43, 46 and 55 kDa, were expressed. Further work focused on the 43 kDa protein because it was expressed at levels detectable by SDS-PAGE and immunoblot analysis. The nucleotide sequence of the 1.8 kbp insert containing the gene encoding this protein was determined. The sequence contained three open reading frames (ORFs). The first ORF (ORF1) did not appear to code for any known protein. The second ORF (ORF2) encoded a protein of 403 amino acids (43,662 Da). The deduced amino acid sequence showed 77% identity (84% similarity) with the tryptophan synthase beta subunit (TrpB) of Salmonella typhimurium and Vibrio parahaemolyticus. The eight conserved regions of TrpB are observed in the P. multocida enzyme, including the conserved lysine (Lys-88) and consensus sequence (GGGSNA) implicated in pyridoxal phosphate binding. The expression and identity of the P. multocida TrpB were confirmed by complementation studies using E. coli W3110 tnaA2 trpB9578. The third ORF (ORF3) consisted of the first 77 nucleotides of the gene encoding the alpha-subunit of tryptophan synthase (trpA), and overlapped the 3'-end of trpB by 14 nucleotides. The deduced amino acid sequence of the 77 nucleotides of the P. multocida TrpA had 68% identity (92% similarity) with the analogous region of TrpA from Klebsiella aerogenes (K. pneumoniae).

Amino Acid Sequence↗
PubMed1996

Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34.

The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or beta-glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobhacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5' deletion analyses of the rpL34 promoter indicated that a 50 bp region located between -179 and -129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3' deletion analyses. A 3' deletion up to -28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to - 104. eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.

Base Sequence↗
PubMed2001

Structure and expression of the Ah receptor repressor gene.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

5' Untranslated Regions↗
PubMed1990

Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.

Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.

Acyltransferases↗
PubMed1999

The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.

A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of alpha-D-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular alpha-glucuronidase (aguA) and a beta-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.

Base Sequence↗
PubMed1991

Cloning of the human erythropoietin receptor gene.

We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5' region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5' of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5' flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5' of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5' to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of a mouse beta 1-adrenergic receptor genomic clone.

A beta 1-adrenergic receptor (beta 1AR) clone, designated Clone 5, was isolated from a BALB/c mouse liver genomic library screened at low stringency with a human brain beta 2 AR cDNA probe. Sequence analysis of Clone 5 revealed a 1,395-bp open reading frame encoding a 464-amino-acid polypeptide. The predicted protein exhibited structural features characteristic of members of the G-protein-coupled receptor family including seven hydrophobic segments corresponding to putative transmembrane domains, a potential N-linked glycosylation site near the amino-terminus, and multiple potential phosphorylation sites in the third cytoplasmic loop and carboxy-terminal cytoplasmic tail. The sequence of the Clone 5-encoded protein was nearly identical to those previously reported for the rat and human beta 1 ARs. Potentially important differences were noted in the third cytoplasmic loop and carboxy-terminal cytoplasmic tail. Reverse transcription-primer extension studies of adult mouse brain RNA demonstrated the predominant transcriptional start site to be 415 nucleotides upstream of the translational start site. A GC-box precedes the transcriptional start site by 40 nucleotides. No consensus TATA or CAAT box sequences were identified in this region. Southern blot analysis of a Chinese hamster x mouse somatic cell hybrid panel and of the progeny of an inter-subspecies backcross mapped the Clone 5-encoded gene to mouse chromosome 19, the localization previously determined for the mouse homolog of the human beta 1AR gene. Binding studies of transient COS-7 transfectants and stable L-cell transfectants confirmed that Clone 5 encodes a beta AR of the beta 1 subtype. A probe derived from Clone 5 selectively hybridized in Northern blot studies to mRNA isolated from adult mouse cerebrum, lung, and heart. These data should serve as the basis for further studies of the regulation and function of the beta 1AR.

Amino Acid Sequence↗
PubMed1998

Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis.

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [3H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467-3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.

Adhesins, Bacterial↗
PubMed1990

Isolation and characterization of the rat plasminogen activator inhibitor-1 gene.

We have reported that glucocorticoids and cyclic nucleotides regulate tissue-type plasminogen activator activity in HTC rat hepatoma cells primarily by modulating plasminogen activator inhibitor (PAI-1) gene expression. To investigate the molecular mechanisms underlying this hormonal regulation, we have cloned the rat PAI-1 gene from an HTC genomic library. The gene is approximately 10.5 kilobases (kb) in size and is comprised of nine exons and eight introns. Comparison of this gene with the human PAI-1 gene (Bosma, P. J., van den Berg, E., Kooistra, T., Siemieniak, D. R., and Slightom, J. L. (1988) J. Biol. Chem. 263, 9129-9141) revealed strict conservation of the exon-intron structure. The start site of transcription is identical to that in the human PAI-1 gene. The complete nucleotide sequence of the rat PAI-1 gene, including 2.4 kb of 5'- and 1 kb of 3'-flanking DNA, has been determined. Two regions in the 5'-flanking sequence of the rat PAI-1 gene show a high degree of similarity to sequences in the upstream region of the human PAI-1 gene: a 60-base pair region from -90 to the TATA box (90% identical), and a more distal sequence located at -753 to -510 (greater than 80% identical). Both of these regions have been shown to be involved in the glucocorticoid regulation of expression of the human PAI-1 gene (van Zonneveld, A.-J., Curriden, S. A., and Loskutoff, D. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5525-5529), although neither region contains the hexamer consensus sequence for glucocorticoid response elements. Five putative glucocofticoid response elements, 100% identical to the hexamer consensus sequence, were identified in the 5'-flanking region of the rat gene, none of which is present in the regions of sequence similarity. Seven putative cyclic AMP response elements that are 75% identical to the octamer consensus sequence are located upstream of the rat gene. There is only one such sequence within the 5'-flanking region of the human gene; however, that sequence, at -61, is 100% conserved in the rat gene.

Amino Acid Sequence↗
PubMed2000

Molecular cloning of ovine connexin44 and temporal expression of gap junction proteins in a lens cell culture.

PURPOSE: The lens plasma membranes of several mammalian species have been shown to contain three different connexin proteins. The goal of this study was to clone the sheep homologue of rat connexin46 identified as sheep connexin44 and to determine the temporal changes in the expression of the three sheep connexin proteins in a lens primary cell culture system. METHODS: A sheep genomic library was screened with a rat lens connexin46 cDNA probe. Lens junctional protein and mRNA levels were determined in a sheep primary cell culture system by Western and Northern blot analyses, respectively. RESULTS: Sheep connexin44, the homologue of rat lens connexin46, was identified as a single-copy gene with a predicted molecular weight of 43,989 Daltons that is contained within a single exon. Northern blot analysis detected a 2.2-kb connexin44 transcript in RNA isolated from lens but not that isolated from heart, kidney, liver, or lung. During the in vitro differentiation of lens epithelial cells from 5 to 20 days in culture, connexin43 mRNA levels declined approximately 75%, whereas connexin49 RNA levels increased approximately 24 fold. The 40% decrease in the level of connexin43 protein and the 21-fold increase in the level of connexin49 protein did not directly correlate with the changes in mRNA levels encoding these proteins during this same period. Although detectable, the amount of connexin44 mRNA and protein remained low throughout the 20-day period during which lens cells were grown in culture. Neither mRNA nor protein encoding MP20 or MP26 transcripts could be detected in even the oldest 20-day lens cultures. CONCLUSIONS: Steady state mRNA levels of sheep connexin43 and connexin49 do not appear to be the only factor regulating the expression of these genes during in vitro differentiation of lens cells in culture. Although a decreased level of expression of connexin43 was accompanied by an increased level of expression of connexin49 over the 20-day period in culture, connexin44 mRNA and protein levels remained low throughout this 20-day period. Overall, these results suggest that these junctional proteins have a unique temporal pattern of expression during differentiation, and this lens primary cell culture system provides a valuable tool to better understand this process.

Animals↗
PubMed1993

Characterization of the promoter-regulatory region and structural organization of E1 alpha gene (BCKDHA) of human branched-chain alpha-keto acid dehydrogenase complex.

We have isolated genomic clones containing the complete exon 1 and the promoter-regulatory region of the E1 alpha gene (BCKDHA) of human branched-chain alpha-keto acid dehydrogenase complex. The cloning was achieved by amplification of a genomic library in the SRB (P2) host strain that allowed the replication of nonstandard DNA structures. The results of this and previous (Dariush, N., Fisher, C. W., Cox, R. P., and Chuang, D. T. (1991) FEBS Lett. 284, 34-38) studies showed that the human E1 alpha gene contains 9 exons, and spans at least 55 kilobases (kb). Exon 1 is 135 bp in length, and contains multiple transcription initiation sites at bases +1, +18, and +22. The complete human E1 alpha cDNA is, therefore, 1,758 bp in length excluding the poly(A)+ tail, and has 27 nucleotides in the 5'-untranslated region. Sequencing of the 5'-flanking region disclosed the absence of a canonical TATA-box in the vicinity of base -30. Several sets of "CAAT" box-like sequences and Sp1 binding-sites are present. Also present are copies of potential AP-2 binding, fat-specific element 1, fat-specific element 2, glucocorticoid-responsive element, and cAMP-responsive element sequences, as well as multiple sets of direct and inverted repeats. The promoter-regulatory region was characterized using deletion constructs and the luciferase reporter assay. The human hepatoma cells (Hep-G2) and Chinese hamster ovary (CHO) cell lines were used as hosts. The results obtained with Hep-G2 cells indicate that the region for high level transcription is located between bases -320 and -115. Extension of the 5'-end of the insert to beyond base -320 markedly reduces promoter activity. The results strongly suggest the presence of inhibitory elements in the region upstream of base -320. Assays in CHO cells show that the region for high level transcription lies between bases -909 and -115. The variation in the region for high level transcription in Hep-G2 and CHO cells may represent cell-type specific differences in the E1 alpha gene promoter function.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs.

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.

Allosteric Regulation↗
PubMed1991

A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation.

Saccharomyces cerevisiae strains carrying vps18 mutations are defective in the sorting and transport of vacuolar enzymes. The precursor forms of these proteins are missorted and secreted from the mutant cells. Most vps18 mutants are temperature sensitive for growth and are defective in vacuole biogenesis; no structure resembling a normal vacuole is seen. A plasmid complementing the temperature-sensitive growth defect of strains carrying the vps18-4 allele was isolated from a centromere-based yeast genomic library. Integrative mapping experiments indicated that the 26-kb insert in this plasmid was derived from the VPS18 locus. A 4-kb minimal complementing fragment contains a single long open reading frame predicted to encode a 918-amino-acid hydrophilic protein. Comparison of the VPS18 sequence with the PEP3 sequence reported in the accompanying paper (R. A. Preston, H. F. Manolson, K. Becherer, E. Weidenhammer, D. Kirkpatrick, R. Wright, and E. W. Jones, Mol. Cell. Biol. 11:5801-5812, 1991) shows that the two genes are identical. Disruption of the VPS18/PEP3 gene (vps18 delta 1::TRP1) is not lethal but results in the same vacuolar protein sorting and growth defects exhibited by the original temperature-sensitive vps18 alleles. In addition, vps18 delta 1::TRP1 MAT alpha strains exhibit a defect in the Kex2p-dependent processing of the secreted pheromone alpha-factor. This finding suggests that vps18 mutations alter the function of a late Golgi compartment which contains Kex2p and in which vacuolar proteins are thought to be sorted from proteins destined for the cell surface. The Vps18p sequence contains a cysteine-rich, zinc finger-like motif at the COOH terminus. A mutant in which the first cysteine of this motif was changed to serine results in a temperature-conditional carboxypeptidase Y sorting defect shortly after a shift to nonpermissive conditions. We identified a similar cysteine-rich motif near the COOH terminus of another Vps protein, the Vps11/Pep5/End1 protein. Preston et al. (Mol. Cell. Biol. 11:5801-5812, 1991) present evidence that the Vps18/Pep3 protein colocalizes with the Vps11/Pep5 protein to the cytosolic face of the vacuolar membrane. Together with the similar phenotypes exhibited by both vps11 and vps18 mutants, this finding suggests that they may function at a common step during vacuolar protein sorting and that the integrity of their zinc finger motifs may be required for this function.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Structural organization of the rat luteinizing hormone (LH) receptor gene.

The luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor gene was isolated from rat liver genomic libraries and spanned at least 75 kilobase pairs from nucleotide -2057 of the 5'-flanking region to the 3'-noncoding end. The structural configuration of the coding region of the LH receptor gene consists of 11 exons separated by 10 introns that are all located within the putative extracellular domain prior to the first transmembrane region. The 5'-noncoding region contains several potential TATA boxes and SP1 promoter binding sites, as well as six palindromic elements, potential intron/exon splice junctions, and two extended open reading frames in frame with the initiation codon. Primer extension studies indicate the presence of multiple transcriptional initiation sites. Truncated forms of the LH/hCG receptor conform to alternative splicing patterns that are consistent either with the deletion of complete exons or alternate acceptor sites within exons. All splice site junctions correspond to known donor and acceptor consensus sequences. Exons 2-8 approximate the regions of the 14 consecutive 20 amino acid repeated motifs present in the LH, thyrotropin-stimulating hormone, and follicle-stimulating hormone receptor cDNAs, with the exception of a six to eight amino acid shift in each motif. Exons 1, 9, 10, and 11, do not conform to this repetitive intronic motif. These exons, however, contain important structural elements including the conserved cysteines (exons 1, 9, 11), the soybean lectin motif (exon 9), the seven-transmembrane domain with cytoplasmic G protein coupling elements (exon 11), and three putative N-linked glycosylation sites (exon 10), consistent with preservation of significant functional domains within single exons. Exon 10 and the beginning of exon 11 along with the lectin motif are unique to the LH receptor and may be involved in specific association with the hormonal ligand. The homologous regions with other members of the glycoprotein receptor family encoded by exons 2-8, and the common amino acid motif that contains 3 conserved cysteines immediately prior to the first transmembrane region, may be involved in common hormonal interactions and in coupling functions, respectively.

Amino Acid Sequence↗
PubMed1997

Characterization of the hamster CYP11B2 gene encoding adrenal cytochrome P450 aldosterone synthase.

A CYP11B2 gene encoding cytochrome P450 aldosterone synthase (P450aldo) was isolated from a hamster genomic library. The gene, which contained 9 exons, was composed of 9,045 bp, of which 3,722 bp were located in the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) and other putative cis elements, previously named Ad1 to Ad6, were identified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nuclear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a new protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis element was specifically retarded by factors in the nuclear extracts of hamster adrenal ZG. We then examined the transcriptional activity of the 5' UTR of the CYP11B2 gene, using chloramphenicol acyltransferase (CAT) as the reporter gene. Ten deletion plasmids were constructed using a modified pCAT vector. Transient transfections of the chimeric reporter constructs into Y1 cells showed that the highest basal promoter activity was obtained with the construct containing up to -134 bp. Increasing the length of the regulatory region of CYP11B2 gene to -167 bp resulted in less than two-thirds of the maximal activity, indicating the probability of putative inhibitory cis elements in this area of the gene. Forskolin stimulated the expression of the reporter gene of deletion plasmids excepting the construct containing only the TATA box, and the highest activity also occurred with the -134 bp construct. TPA had no stimulatory effects on any of the constructs, and interestingly it slightly inhibited CAT activity. In contrast to TPA, staurosporine, an inhibitor of the PKC pathway, stimulated CAT activity. To conclude, the promoter region of the hamster CYP11B2 gene transfected in Y1 cells is responsive to forskolin, indicating that the gene is controlled by the PKA signaling pathway. Paradoxically, staurosporine, but not TPA, stimulates the promoter activity of the CYP11B2 gene, indicating that PKC might, at least in Y1 cells, act as a negative regulator on the aldosterone synthase promoter. Moreover, a new cis element was shown to exert a negative effect on basal as well as on stimulated activities of the hamster promoter CYP11B2 gene.

Animals↗