Biomedical subjects

W J Rutter

Publications and source records attributed to W J Rutter.

At least 145 records · Page 8Linked to original sources

PubMed1984

Structure of two related rat pancreatic trypsin genes.

A family of approximately 10 trypsin genes was detected in a rat genomic library by hybridization and in vivo recombination techniques using cloned rat pancreatic trypsin I and II cDNAs as probes. Two separate clones containing the entire trypsin I gene and most of the trypsin II gene were sequenced. Four introns split the trypsin I coding sequence. The positions of the first three introns of the trypsin II gene are identical with those in the trypsin I gene (the fourth intron was not present in the trypsin II clone). The coding regions of the two genes are 88% homologous; the 5'-noncoding regions are 92% homologous, whereas the 3'-noncoding regions share 66% identity. In contrast, the proximal 5'-flanking regions from -1 to -500 which may contain the elements controlling gene expression are less than 30% conserved overall, but segments of approximately 70% homology can be discerned in this region. Some of these sequences are homologous to sequences found in the chymotrypsin and elastase genes. More distal upstream sequences (-500 to -2500) and the intervening sequences show no evident sequence homology (less than 20%). Unique sequences containing homopolymeric purine/pyrimidine repeats are found 2.5 kilobases upstream from the start of transcription of the trypsin I gene and within the second and third introns of the trypsin II gene. The nucleotide homologies as well as the similarities of intron positions of the two trypsin genes to those of other serine protease genes clearly support an evolutionary relationship between members of this gene family.

Animals↗

PubMed1984

Structure of the two related elastase genes expressed in the rat pancreas.

We have isolated and characterized rat genomic DNA fragments bearing the two secretory elastase genes that are expressed in the exocrine pancreas. The complete exonic sequences for each of the genes as well as considerable intronic and flanking sequences are reported. Each elastase gene is interrupted by seven intervening sequences which are located at corresponding positions within the two genes, with one exception: the third intron of the elastase II gene has shifted one codon in the 5' direction. The placement of introns within the amino acid coding domains in part may reflect the formation of the progenitor serine protease gene by the duplication of an exon encoding a characteristic polypeptide structure comprising three beta sheets. The activation peptides of the zymogens and the signal peptides, which form discrete functional domains in the protein precursors, are encoded by separate exons. In addition to the TATAA box, the two genes share considerable sequence similarity in the 5'-proximal flanking regions (up to approximately 450 base pairs upstream); however, a number of gaps must be introduced to optimize the sequence alignment. The similarities are largely confined to six oligonucleotide regions with greater than 70% sequence conservation. The elastase I gene has a perfect repeating copolymer (GT)24 located 427-379 nucleotides upstream from the start of transcription. The elastase II gene has a similar GT-rich region (52/55 G or T) located 384-330 nucleotides upstream. Comparison of the 5'-flanking regions of the two elastase genes with those of pancreatic chymotrypsin and trypsin I and II reveals that one of the six conserved oligonucleotide regions is generally conserved for these genes as well. This conserved region contains putative enhancer core sequences.

Amino Acid Sequence↗

PubMed1984

Isolation and sequence of a rat chymotrypsin B gene.

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence analysis of a proline-rich protein cDNA and peptide homologies of rat and human proline-rich proteins.

Plasmids containing cDNAs for proline-rich proteins have been isolated from a cDNA library prepared from parotid glands of isoproterenol-treated rats. The nucleotide sequence of one of these cDNAs (pRP33) has been determined, and it encodes an acidic proline-rich protein. The N-terminal 13 amino acids are highly hydrophobic and may be part of a signal peptide. The next 66 amino acids constitute an acidic domain which is interrupted by a short (8 residue) basic region. The sequence beginning at proline 80 contains six tandemly repeated regions of 18 to 19 amino acids in length with the prototype sequence PPPQGGPQXPPQPGXPQG. Two pairs of these repeats (amino acid residues 116 to 152 and 153 to 189) are 93% homologous in nucleotide sequence. The high homology in both nucleotide and amino acid sequences in the repeat region suggests that this protein may have evolved by duplication of internal portions of a progenitor gene.

Amino Acid Sequence↗

PubMed1984

Sequence of the human somatostatin I gene.

Two human genomic DNA fragments containing alleles for the gene coding for somatostatin I were isolated and sequenced. This gene contains a single intron that interrupts the coding sequence in the propeptide portion of the somatostatin moiety. The site of initiation of transcription of the gene was located by transcription experiments in HeLa cell extracts, and the putative regions for controlling the initiation of transcription were identified.

Alleles↗

PubMed1984

Chromosomal assignments of human genes for serine proteases trypsin, chymotrypsin B, and elastase.

The genes for the serine proteases trypsin, chymotrypsin B, and elastase were chromosomally assigned in man using cDNA probes that have been isolated from a rat pancreatic cDNA library. DNA from human X rodent somatic cell hybrids was cleaved with BamHI or EcoRI and analyzed by Southern filter hybridization methods for the segregation of the genes for trypsin-1 (TRY1), chymotrypsin B (CTRB), and elastase-1 (ELA1). TRY1 was assigned to human chromosome 7q22----qter, CTRB to chromosome 16, and ELA1 to chromosome 12. Although the three genes are members of the same gene family, they are dispersed over different chromosomes.

Biological Evolution↗

PubMed1984

Chromosomal assignments of genes for trypsin, chymotrypsin B, and elastase in mouse.

The mouse genes for the serine proteases trypsin (Try-1), chymotrypsin B (Ctrb), and elastase (Ela-1) were chromosomally assigned using Southern blot hybridization of mouse X Chinese hamster cell hybrid DNA. cDNA probes for the three genes were hybridized to cell hybrid DNA cleaved with BamHI or HindIII and the segregation of Try-1, Ctrb, and Ela-1 was correlated with the segregation of mouse chromosomes. Try-1 is located on chromosome 6, Ctrb is on chromosome 8, and Ela-1 is on chromosome 15. The three genes fall into three syntenic groups that are conserved in the mouse and human genomes.

Animals↗

PubMed1984

Transcription of the hepatitis B surface antigen gene in cultured murine cells initiates within the presurface region.

Cloned hepatitis B virus (HBV) DNA directs the synthesis of the viral surface antigen (HBsAg) when introduced into mouse L cells by DNA transformation. We have used recombinants between the Rous sarcoma virus long terminal repeat and subgenomic fragments of HBV DNA to localize regions of the HBV genome required for HBsAg expression. Examination of HBV-specific RNA from such transformants indicates that transcription initiates at three distinct sites (153, 163, and 183 nucleotides upstream from the translation initiation codon for mature HBsAg). Thus in these cells, a large segment of the presurface reading frame is not represented in HBsAg mRNA. The termination site of this RNA lies within the coding sequences for the viral core antigen, some 1,094 +/- 10 base pairs downstream from the TAA stop codon for HBsAg. Two additional open reading frames are present in the resultant unspliced HBsAg RNA.

Animals↗

PubMed1984

Cloning and analysis of integrated hepatitis virus sequences from a human hepatoma cell line.

We report here the isolation by molecular cloning and the analysis by heteroduplex and restriction enzyme mapping of seven distinct DNA fragments containing hepatitis B virus (HBV) sequences from genomic DNA of the PLC/PRF/5 human liver carcinoma cell line (the Alexander cell). No intact full-length HBV genomes were present. Three different patterns of organization of HBV fragments were detected. These included two linear fragments without detectable rearrangement, three other HBV fragments with internal deletions, and two HBV fragments containing long inverted duplications. HBsAg sequences are preferentially included in the integrated fragment, whereas the core gene is preferentially eliminated. Several of the integrated HBV fragments might act as templates for the synthesis of functional HBsAg mRNA, whereas only one clone could produce a full core antigen transcript.

Base Sequence↗

PubMed1984

A complementary DNA sequence that predicts a human pancreatic amylase primary structure consistent with the electrophoretic mobility of the common isozyme, Amy2 A.

We report the nucleotide sequence of mRNA for the common electrophoretic isozyme of human pancreatic alpha-amylase, Amy2 A. The sequence was derived from a nearly full-length complementary DNA (cDNA) isolated from a cloned cDNA library. The relatively short 5' untranslated region (15 nucleotides) was determined by primer-extension sequencing. The human Amy2 messenger codes for a 511-residue preamylase polypeptide. An amino-terminal signal peptide of 15 amino acids with an Ala X Gln cleavage site is proposed based on homology to mouse, dog and hog amylases. The Amy2 A mRNA sequence differs from a recently reported human Amy2 sequence. Differences were found at 31 nucleotide positions. The alpha-amylase proteins predicted by the two mRNAs differ at 17 amino acid positions. Relative to the known sequences of other mammalian amylases, most of the differences between the two human Amy2 sequences appear to have occurred as substitutions in the sequence reported by Nakamura et al. (1984). These substitutions predict a protein with a substantially greater net negative charge than that of Amy2 A. We suggest that the two sequences may represent either divergent Amy2 alleles or the expression of non-allelic pancreatic amylase genes.

Amino Acid Sequence↗

PubMed1983

Structure of a mouse submaxillary messenger RNA encoding epidermal growth factor and seven related proteins.

The structure of the messenger RNA (mRNA) encoding the precursor to mouse submaxillary epidermal growth factor (EGF) was determined from the sequence of a set of overlapping complementary DNA's (cDNA). The mRNA is unexpectedly large, about 4750 nucleotide bases, and predicts the sequence of preproEGF, a protein of 1217 amino acids (133,000 molecular weight). The EGF moiety (53 amino acids) is flanked by polypeptide segments of 976 and 188 amino acids at its amino and carboyxl termini, respectively. The amino terminal segment of the precursor contains seven peptides with sequences that are similar but not identical to EGF.

Amino Acid Sequence↗

PubMed1983

Splice junctions: association with variation in protein structure.

A comparison between eukaryotic gene sequences and protein sequences of homologous enzymes from bacterial and mammalian organisms shows that intron-exon junctions frequently coincide with variable surface loops of the protein structures. The altered surface structures can account for functional differences among the members of a family. Sliding of the intron-exon junctions may constitute one mechanism for generating length polymorphisms and divergent sequences found in protein families. Since intron-exon junctions map to protein surfaces, the alterations mediated by sliding of these junctions can be effected without disrupting the stability of the protein core.

Amino Acid Sequence↗

PubMed1983

Expression of the human insulin gene in an alternate mammalian cell and in cell extracts.

The functional regions of the human insulin gene have been characterized by in vitro and in vivo experiments. The locations of the insulin promoter and cap site have been predicted from the sequence of the gene, and these assignments are consistent with the results of in vitro transcription experiments using HeLa cell extracts. For studies of intracellular expression, the human insulin gene including its promoter and two intervening sequences was inserted into the late region of the virus, SV40, in a construction that eliminates the major splice junction of the late SV40 region. Permissive CV1 monkey kidney cells were infected with this recombinant and a complementing helper virus. During the course of the infection, insulin gene transcripts accumulate at about one-third the level of SV40 late transcripts originated from the same promoter. Analysis of the RNAs containing insulin sequences showed that the insulin introns are excised with precision. Furthermore, the insulin promoter (cap site) and polyadenylation sites are recognized but are less efficiently employed than the viral late promoter and terminator. The mRNA is translated and immunoreactive human proinsulin is secreted into the extracellular medium.

Animals↗

PubMed1983

Expression of the human insulin gene and cDNA in a heterologous mammalian system.

The human insulin gene or the corresponding cDNA has been inserted into the early region of a simian virus 40 vector in which all SV40 splice junctions were deleted while the early promoter and polyadenylation regions remained intact. The expression of insulin-coding sequences was tested in permissive monkey COS cells. The insulin cDNA was transcribed from the early promoter to produce a stable polyadenylated RNA which was translated, and immunoreactive human proinsulin accumulated in the medium. Thus RNA splicing is not obligatory for insulin expression in this system. The genomic insulin transcript was also initiated from the SV40 promoter and terminated at the SV40 polyadenylation site. S1 endonuclease mapping revealed that the transcript is processed via two alternative splicing pathways within the insulin gene. About one-third of the total transcripts are processed normally with removal of the two insulin-specific introns. This transcript is apparently translated normally since immunoreactive proinsulin accumulates in the medium. About two-thirds of the transcripts are processed via an alternative splicing pathway involving a new splice acceptor site located within the coding region of the insulin gene. This results in a codon frameshift such that translation would produce a novel chimeric peptide containing the insulin NH2-terminal B chain, but a different COOH terminus containing human and SV40 sequences. A peptide of the predicted size is detected in the COS cell extract.

Animals↗

PubMed1983

Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor.

Nerve growth factor (NGF) is a polypeptide that enhances survival, nerve fibre outgrowth and neurotransmitter biosynthesis in sympathetic and sensory neurones. Administration of antibodies against NGF to developing animals leads to atrophy of the sympathetic system. NGF is not normally detectable in innervated tissues but ablation of the innervating neurones leads to the production of measurable NGF in the target tissue. After transplantation of the denervated tissue, reinnervation occurs, then NGF decreases to undetectable levels. Thus NGF seems to act as a neurotrophic messenger and its level is regulated by innervating neurones. Because of the minute levels present it is very difficult to study NGF biosynthesis in innervated tissue. However, NGF can be isolated from male mouse submaxillary glands, where it exists in inexplicably high levels. Its amino acid sequence has been determined, and the synthesis of NGF and its larger precursors has been demonstrated in cultured submaxillary glands. We report here the nucleotide sequence of a submaxillary cDNA encoding the mouse NGF precursor (preproNGF). In contrast to previous suppositions the NGF moiety is situated near the carboxyterminus of the polyprotein precursor. It is flanked at the amino-terminus by 187 amino acids which may be cleaved at dibasic residues to generate three peptides; there are only two additional amino acids at the carboxy-terminus.

Amino Acid Sequence↗

PubMed1983

Isolation and characterization of a rat amylase gene family.

Portions of at least nine distinct rat amylase genes or pseudogenes have been isolated. Cloned rat genomic DNA fragments containing complete or major portions of seven of these have been examined by heteroduplex analysis and fall within two separate groups based on their degree of homology. Four gene sequences comprising one of these groups are closely related to pancreatic amylase mRNA. The other group shows significant nonhomology to both pancreatic and parotid amylase cDNAs and may represent an additional gene type(s). All of the cloned amylase gene sequences are found in rat genomic DNA. Additional amylase sequences which have not yet been cloned are also detected. Comparison of DNA from individual Sprague-Dawley rats by Southern blotting techniques indicates allelic variation at multiple amylase loci.

Amylases↗

PubMed1983

Effects of dexamethasone and 5-bromodeoxyuridine on protein synthesis and secretion during in vitro pancreatic development.

Protein synthesis and secretion during in vitro pancreatic development and after treatment with the glucocorticoid dexamethasone and the thymidine analog 5-bromodeoxyuridine (BrdU) was monitored using two-dimensional gel electrophoresis. At 14 days gestation, the synthesis of more than 200 proteins and the secretion of a complex set of proteins was detected. The relative rate of synthesis and secretion of the majority of this set of proteins decreased dramatically during development; after 6 days of culture most were no longer detected. In contrast, the synthesis and secretion of pancreas-specific exocrine proteins amylase, a Sepharose binding protein (protein 2), and chymotrypsinogen first detected after one day in culture, increased throughout the 6-day culture period. Other pancreatic digestive (pro)enzymes normally found in the adult such as the basic form of chymotrypsinogen, lipase, ribonuclease, and trypsinogen were not detected during the culture period. Thus at least two distinct regulatory events are involved in the expression of the exocrine genes during development. Dexamethasone treatment during the 6-day culture period selectively increased the synthesis of amylase and several other minor secretory proteins. BrdU treatment caused major changes in the protein synthetic and secretory patterns of the pancreas as well as in morphogenesis. BrdU treated pancreases showed greatly reduced synthesis of amylase, protein 2, and chymotrypsinogen and prolonged synthesis of many proteins normally detected only at early stages of pancreatic development. BrdU treatment also stimulated the secretion of a set of proteins ostensibly associated with duct cells. Thus, BrdU specifically alters the developmental program of the pancreas.

Age Factors↗

PubMed1983

Tubulin genes are tandemly linked and clustered in the genome of trypanosoma brucei.

We have isolated cDNA and genomic clones containing alpha- and beta-tubulin genes from Trypanosoma brucei. Each clone has been mapped, and the identity of the tubulin genes has been established by cross-hybridization with cloned chicken tubulin genes and by hybridization-selection and translation of trypanosome tubulin mRNA. In contrast with the dispersed organization of tubulin genes in other organisms, trypanosome alpha- and beta-tubulin genes are physically linked and clustered in tandem repeats of approximately 13-17 copies per haploid genome of alternating alpha- and beta-tubulin sequences.

Animals↗