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At least 1,243 records · Page 69Linked to original sources
PubMed1994

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the human prostatic adenocarcinoma cell line LNCaP.

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Adenocarcinoma↗
PubMed1996

Cloning, sequencing and functional analysis of a truncated cDNA encoding red deer prolactin receptor: an alternative tyrosine residue mediates beta-casein promoter activation.

This study reports the isolation and in vitro characterisation of a truncated cDNA encoding the red deer long form prolactin receptor. The cDNA sequence predicts a protein of 557 amino acids which differs from the rat sequence by a 3' truncation of the cytoplasmic domain located 34 residues before the stop codon. The deer sequence shares the regions of homology which are important for maintenance of structural and functional integrity, high affinity binding and signal transduction. However, the truncated deer receptor lacks the most C-terminal tyrosine residue in the intracellular domain which is believed to be essential for activation of the beta-casein promoter. Transfection studies of the cervine cDNA into human 293 fibroblast cells confirmed the expression of a receptor that has high affinity binding to ovine prolactin (Ka = 0.65 x 10(9)M(-1) and Bmax = 548.6 fmol/mg protein). Co-transfection of CHO cells with expression vector encoding the cervine prolactin receptor cDNA along with a fusion gene containing the promoter region of beta-casein followed by beta-luciferase coding sequence led to 8.13 +/- 0.13-fold induction of luciferase enzyme activity in the presence of 400 ng/ml ovine prolactin. This was comparable to fold induction observed with the wild type long form rat prolactin receptor (6.37 +/- 0.48); macaque growth hormone receptor was without effect. Western blot analysis demonstrated tyrosine phosphorylation of the cervine receptor and the associated kinase Jak2 following stimulation with prolactin. This confirms that the cervine cDNA although truncated is fully functional and that Jak2 and an alternative tyrosine residue in the intracellular domain are involved in the signalling pathway leading to activation of the beta-casein promoter. Northern blot analysis provides evidence that the prolactin receptor in the liver is encoded by transcripts of approximately 2.5 and 3.5 kb. Comparison of Northern blots of different deer species suggests that the receptor is conserved amongst the Cervidae. Northern blot analysis of red deer testis suggests that this species expresses a second form of the receptor, encoded by a transcript of 1.7 kb, which may correspond to a smaller receptor form or a binding protein.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of zinc finger encoding genes from the tumoral exocrine pancreatic cell line AR42J.

Zinc finger transcription factors are DNA-binding proteins that are known to determine the identity of cells by regulating cell-specific gene expression. In addition, mutations in some members of this family have been found to be associated with several neoplasias, including Wilms' tumor and leukemias. Because the mechanisms that regulate normal, as well as neoplastic, pancreatic cell differentiation are poorly understood, we are searching for pancreas-enriched transcription factors that may determine the identity of pancreatic cells. Towards this end, we have used the polymerase chain reaction and degenerate primers against the highly conserved (Cys2-His2) zinc finger domain to amplify novel transcription factor encoding cDNAs from the well-characterized pancreatic acinar cell line AR42J. Using this approach, we have identified 17 different zinc finger encoding cDNAs (AZF-1 to -17). Sequence analysis shows that all of these clones encode for different zinc finger peptides which share the consensus DNA binding motif with the Drosophila transcription factor krüppel. As a first step in the characterization of these genes, the purified PCR products were used to determine their spatial pattern of expression by northern blot analysis. Using these techniques, we find that numerous zinc finger encoding genes are expressed in AR42J acinar cells as well as in normal adult rat pancreas and suggest that they may play a role as transcription factors in exocrine pancreatic cells.

Age Factors↗
PubMed1996

A novel profile of expressed sequence tags for zinc finger encoding genes from the poorly differentiated exocrine pancreatic cell line AR4IP.

Genes encoding for C2H2 zinc finger proteins are known to regulate normal cell proliferation and differentiation and have often been found to be mutated in different forms of cancer. We are interested in understanding the role of these genes as regulators of cell proliferation and differentiation in the exocrine pancreas. Therefore, we have generated expressed sequence tags (ESTs) encoding pancreas-enriched zinc finger peptides using the polymerase chain reaction and hybridization techniques [Adams, M.D. et al. (1991) Science, 252, 1651-1656]. Here we report the primary structure and expression pattern of 18 different zinc finger-encoding cDNAs (DZF-1-18) from the azaserine-derived tumoral cell line AR4IP which displays a poorly differentiated phenotype. Sequence analysis shows that all of these clones encode peptides which share the consensus DNA-binding motif with the Drosophila zinc finger transcription factor krüppel. High stringency Northern blot analysis shows that eight different zinc finger transcripts are expressed at high levels in normal adult rat pancreas and therefore constitute good candidates to play a role as transcription factors in exocrine pancreatic cells.

Animals↗
PubMed1992

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus.

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.

Amino Acid Sequence↗
PubMedDate not supplied

The nucleotide sequence of the gene encoding protein IVa2 in human adenovirus type 7.

The nucleotide sequences of cloned DNA segments encoding the IVa2 gene from Ad7 and a portion of Ad12 (group B and group A human adenoviruses, respectively) have been determined. When compared to Ad5, a group C adenovirus, these sequences have been found to be 80% homologous. Most changes are transitions or transversions. This high degree of nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded from these genes; most nucleotide changes occur at the third position in the codon. The predicted polypeptide contains 448 amino acids and has a calculated Mr-value of 50700. The positions of the 5' end of the mRNA and of the donor and acceptor splice sites of Ad7 and Ad12 can be inferred by analogy to those of Ad5. A long open reading frame starting upstream from the IVa2 gene overlaps the N-terminal portion of the polypeptide but is encoded in a different reading frame. Within this overlapping region, the long open reading frame is more conserved in amino acid sequence than is the presumed IVa2 polypeptide, suggesting that evolutionary pressure was exerted on the longer protein, a product of viral early region 2B. The high degree of conservation of this E2B region within the overlapping segment suggests that its activities must be more important for adenovirus infection than are the functions encoded in the amino-terminus of the IVa2 gene.

Adenoviruses, Human↗
PubMed1986

Ribosomal proteins are encoded by single copy genes in Dictyostelium discoideum.

Five recombinant plasmids which encode ribosomal proteins (r-proteins) from Dictyostelium discoideum have been isolated. Poly(A) + RNA was size-fractionated by preparative agarose gel electrophoresis and a fraction encoding proteins of less than 35 kDa was used to construct a cDNA library in the plasmid vector pBR322. Individual clones from the library were screened by hybrid-selected translation and those encoding r-proteins were identified by co-migration of the translation products in two-dimensional gel electrophoresis with marker proteins purified from Dictyostelium ribosomes. Initial characterization using the five cDNA plasmids indicates that these r-proteins are encoded by single copy genes and that they are not tightly clustered in the genome.

Cloning, Molecular↗
PubMed1987

Structure and nucleotide sequence of the region encoding the mobilization proteins of plasmid CloDF13.

Mobilization of the non-conjugative plasmid CloDF13 requires both gene products of a conjugative plasmid and CloDF13-encoded information. About 30% of the CloDF13 genome is involved in plasmid transfer. The CloDF13 mobilization region comprises sequences acting in cis (bom, basis of mobilization) as well as protein-coding sequences (mob). Here we present the nucleotide sequence of the genes encoding the CloDF13 mobilization proteins. We confirmed the previous genetic data that the plasmid CloDF13 encodes two proteins involved in plasmid mobilization. The information for these proteins, designated B and C having Mrs of 57,890 and 15,870, respectively, is located within one operon directed by a promoter at 94% of the CloDF13 genome. The gene encoding the smaller protein is located distally within this operon. Transcription proceeds counter-clockwise and is terminated beyond gene C, although it can not be excluded that attenuation of the transcript occurs in the intergenic region. The role of the CloDF13 mobilization proteins in plasmid transfer will be discussed.

Amino Acid Sequence↗
PubMed1989

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus.

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3'. The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3'-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the Erwinia chrysanthemi gene encoding 2-keto-3-deoxygluconate permease.

The phytopathogenic bacterium Erwinia chrysanthemi produces a group of pectolytic enzymes able to depolymerise the pectic compounds in plant cell walls. The resulting tissue maceration is known as soft rot disease. The degraded pectin products are transported by 2-keto-3-deoxygluconate permease into the bacterial cell, where they serve as carbon and energy sources. This H+ coupled transport system is encoded by the kdgT gene; we report the nucleotide sequence of kdgT. It is encoded by an open reading frame (ORF) of 1194 bp, which is preceded by an Escherichia coli-type promoter region. The ORF encodes a protein with 398 amino acid (aa) residues and a predicted Mr of 48,550. As would be expected for a membrane protein, it is very hydrophobic, containing 63% nonpolar aa. However, the kdgT gene has no apparent evolutionary relationship to other genes encoding sugar transport proteins, such as lacY, melB or the E. coli citrate transport gene. Southern hybridization experiments indicate a strong homology between the Er. chrysanthemi and E. coli kdgT genes; there is also a second region on the E. coli chromosome with homology to kdgT. The kdgT gene is located near the ade-377 marker on the Er. chrysanthemi chromosome (equivalent to the region between 20 and 30 min in E. coli), whereas the E. coli kdgT gene is located at 88 min. Thus, these two enterobacteria show some significant differences in their genomic organization.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of an actin-encoding gene from Hydra attenuata: structural characteristics and evolutionary implications.

We have determined the complete nucleotide sequence of an actin-encoding gene from Hydra attenuata as well as partial sequences of cDNA clones from two additional actin-encoding genes. The gene from the genomic clone contains a single intron, and has promoter and polyadenylation signals similar to those found in other species. The hydra genome has a very A + T-rich base composition (71%). This is reflected in the codon usage of the actin-encoding genes, which is strongly biased towards codons having A or T in the third position. The hydra actin-encoding gene family consists of three or more transcribed genes, two of which are very closely related to each other and probably arose by a recent gene duplication. Hydra actin, like other invertebrate actins, is more similar to the non-muscle isotypes of vertebrates than to the vertebrate muscle actins. Hydra actin is more similar to animal actins than to those of plants or fungi, which is consistent with the view that all metazoans arose from a single protist ancestor.

Actins↗
PubMed1989

Cloning of Schizosaccharomyces pombe genes encoding the U1, U2, U3 and U4 snRNAs.

Schizosaccharomyces pombe contains a group of five relatively abundant small nuclear RNAs (snRNAs) which are immunoprecipitated by human autoimmune antibodies of Sm serotype. The S. pombe RNAs hybridise to probes specific for human U1, U2, U4, U5 and U6 and in each case are similar in size to the human species. A further group of snRNAs from S. pombe are precipitated by antibodies against U3 containing ribonucleoprotein; the most abundant of these species hybridises to a probe specific for human U3. We have cloned the genes encoding U1, U2, U3 and U4 from S. pombe, together with that encoding another abundant snRNA, previously designated SPU43. U2 and U4 are encoded by single-copy genes, while two genes encode U3. The latter are not clustered, since a chromosomal Southern transfer shows them to lie on different chromosomes.

Cell Fractionation↗
PubMed1989

Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli.

A 6-kb fragment of DNA, which complemented defects in the alcohol dehydrogenase (ADH)-encoding gene (adhE) of Escherichia coli, was cloned into a multicopy vector. Both ADH and coenzyme-A-linked acetaldehyde dehydrogenase (ACDH) activities were encoded by the plasmid, pHIL8. The adhE gene was identified as an open reading frame of 891 codons encoding an Mr 96,008 protein (minus the initiating methionine). Codon usage analysis indicates that adhE should be highly expressed. This gene shows no significant homology to any previously sequenced ADH-encoding gene.

Alcohol Dehydrogenase↗
PubMed1989

Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from Alcaligenes eutrophus.

Two DNA fragments encoding the chromosomal and plasmid copies of the gene (cfxP) encoding phosphoribulokinase (PRK) from the chemoautotrophic bacterium Alcaligenes eutrophus, were sequenced and found to be highly homologous. The gene (cfxF) of another Calvin cycle enzyme, fructose-1,6-bisphosphatase (FBPase), was identified as terminating immediately upstream of cfxP, but was not completely contained on both fragments. A hypothetical, also incompletely contained, open reading frame starts closely downstream from cfxP. Genes cfxF, cfxP, and the third hypothetical gene seem to belong to the same operon. The cfxP genes encode highly homologous PRK isoenzyme subunits consisting of 292 aa residues with calculated Mrs of 33 319 (chromosomal PRKc) and 33 164 (plasmid-encoded PRKp). There is little overall sequence similarity between the bacterial and plant (spinach) PRK, apart from some structural motifs.

Alcaligenes↗
PubMed1990

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar.

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris.

The nucleotide sequence of the gene (engXCA) encoding the major extracellular endoglucanase (ENGXCA) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X. c. campestris) was determined and compared with the N-terminal amino acid (aa) sequence of the purified enzyme. An open reading frame of 1479 bp encoding 493 aa was identified, of which the N-terminal 25 aa represent a potential signal peptide. Determination of the exact position of a Tn5 insertion within engXCA, which did not reduce the encoded enzyme activity, indicated that the C-terminal region of the protein is not crucial for ENGXCA activity. Comparison of the complete deduced aa sequence with those deduced from other endoglucanase- and exoglucanase-encoding genes revealed a region with a high degree of homology, located towards the C terminus of the protein. These data indicate that the X. c. campestris ENGXCA may have a domain structure similar to that of many other bacterial and fungal cellulolytic enzymes. Hydrophobic cluster analysis was performed on the deduced aa sequence. Comparison of this analysis with those of 30 other cellulase sequences belonging to six different families indicated that the X. c. campestris enzyme can be classified in family A. The two aa residues which had previously been identified as 'potentially catalytic' within this family of cellulases, are conserved in the X. c. campestris ENGXCA.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of the locus encoding the large and small subunit genes of the periplasmic [NiFe]hydrogenase from Desulfovibrio fructosovorans.

The genetic locus encoding the periplasmic [NiFe]hydrogenase (Hyd) from Desulfovibrio fructosovorans was cloned and sequenced. The genes of this two-subunit enzyme have an operon organization in which the 0.94-kb gene encoding the small subunit precedes the 1.69-kb gene encoding the large subunit. A Shine-Dalgarno sequence is centered at -9 bp from the ATG of both subunits. The possible presence of another open reading frame downstream from the large-subunit-encoding gene is considered. The N-terminal sequence of the large 61-kDa subunit deduced from the nucleotide sequence is in perfect agreement with the results of the amino acid (aa) sequence determined by Edman degradation. A 50-aa leader peptide precedes the small 28-kDa subunit. The aa sequence of the enzyme shows nearly 65% homology with the [NiFe]Hyd aa sequence of Desulfovibrio gigas. Comparisons with a large range of Hyds from various bacterial species indicate the presence of highly conserved Cys residues, the implications of which are discussed from the point of view of nickel atom and cluster accommodation.

Amino Acid Sequence↗
PubMed1991

Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium.

A Phanerochaete chrysosporium BKMF1767 genomic library, constructed in the BamHI site of vector YRp12, was screened with the lignin peroxidase(LIP)-encoding cDNAs, CLG4 and CLG5, that have been shown to encode LIP2 (previously H2) and LIP6 (previously H10), respectively. Six distinct LIP genomic clones, designated pGLG1, pGLG2, pGLG3, pGLG4, pGLG5, and pGLG6, were isolated in this study. Probe CLG4 hybridized only to pGLG1. Probe CLG5 gave intense hybridization to pGLG2 and weaker hybridization to clones pGLG3 through pGLG6, but showed little or no hybridization to pGLG1. These results, in agreement with previous biochemical results, indicate the existence of LIP gene subfamilies. The limits and transcriptional orientation of the LIP gene in each clone were determined. The sequence data showed that pGLG2 contains the LIP6 gene, which encodes a protein identical in amino acid (aa) composition to that encoded by CLG5. It contains a leader sequence of 27 aa and a mature protein of 344 aa (Mr 36,607). Archetypal TATA-box-like and CAAT-box-like sequences in the 5'-noncoding region are located 51 and 97 nt upstream from the cDNA start point, respectively. S1 nuclease analysis of the 5' region of LIP6 revealed two transcription start points 8 nt apart downstream from the TATA box. Comparison of the sequence of LIP6 with its corresponding cDNA CLG5 showed that the gene contains nine small introns which range in size from 50 to 62 bp. These introns contained consensus splice junction sequences similar to those reported in other fungal and yeast introns.

Amino Acid Sequence↗