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L Villa-Komaroff

Publications and source records attributed to L Villa-Komaroff.

52 records · Page 3Linked to original sources
PubMed1987

Alternative 5' exons either provide or deny an initiator methionine codon to the same alpha-tubulin coding region.

The primary structures of two overlapping novel alpha-tubulin cDNA clones isolated from a Macaca fascicularis testis cDNA library and the corresponding human gene are presented. Although the general structure of the human gene conforms to that of previously described mammalian alpha-tubulin genes, there is a surprising difference: the ATG initiator codon is conspicuously absent. The macaque testis cDNA similarly lacks the initiator methionine, but otherwise encodes a variant alpha-tubulin isotype precisely conserved in the human gene. RNA blot analysis in the macaque, using a 3' untranslated region probe, revealed the existence of two additional related transcripts expressed in every tissue examined except the adult testis. Sequence comparisons indicate that the 2.0 kb testis transcript and one of the additional transcripts result from differential transcription of the same gene. The two transcripts differ only at the 5' end as a result of the recruitment of different 5' exons. Curiously, the 5' exon utilized outside the testis encodes an initiator methionine in the expected location.

Amino Acid Sequence↗
PubMed1987

Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor.

Cloned cDNAs encoding neurotensin were isolated from a cDNA library derived from primary cultures of canine enteric mucosa cells. Nucleotide sequence analysis has revealed the primary structure of a 170-amino acid precursor protein that encodes both neurotensin and the neurotensin-like peptide neuromedin N. The peptide-coding domains are located in tandem near the carboxyl terminus of the precursor and are bounded and separated by the paired, basic amino acid residues Lys-Arg. An additional coding domain, resembling neuromedin N, occurs immediately after an Arg-Arg basic amino acid pair located in the central region of the precursor. Additional amino acid homologies suggest that tandem duplications have contributed to the structure of the gene. RNA blot analysis, using the cloned cDNA probe, has revealed several mRNA species ranging in size from 500 to 980 nucleotides in the canine enteric mucosa. In contrast, a single RNA species of 1500 nucleotides was detected in bovine hypothalamus poly(A)+ RNA. The ability of the canine probe to cross-hybridize with bovine mRNA suggests that this probe can be used to isolate neurotensin/neuromedin N genes from other mammalian species.

Amino Acid Sequence↗
PubMed1987

Tissue-specific expression of insulin-like growth factor II mRNAs with distinct 5' untranslated regions.

We have used RNA from human hypothalamus as template for the production of cDNAs encoding insulin-like growth factor II (IGF-II). The prohormone coding sequence of brain IGF-II RNA is identical to that found in liver; however, the 5' untranslated sequence of the brain cDNA has no homology to the 5' untranslated sequence of the previously reported liver cDNAs. By using hybridization to specific probes as well as a method based on the properties of RNase H, we found that the human IGF-II gene has at least three exons that encode alternative 5' untranslated regions and that are expressed in a tissue-specific manner. A probe specific to the brain cDNA 5' untranslated region hybridizes to a 6.0-kilobase transcript present in placenta, hypothalamus, adrenal gland, kidney, Wilms tumor, and a pheochromocytoma. The 5' untranslated sequence of the brain cDNA does not hybridize to a 5.3-kilobase transcript found in liver or to a 5.0-kb transcript found in pheochromocytoma. By using RNase H to specifically fragment the IGF-II transcripts into 3' and 5' fragments, we found that the RNAs vary in size due to differences in the 5' end but not the 3' end.

Chromosome Mapping↗
PubMed1986

Characterization of the two nonallelic genes encoding mouse preproinsulin.

We have cloned and sequenced the two mouse preproinsulin genes. The deduced amino acid sequences of the mature mouse insulins are identical to the published protein sequences. However, the nucleotide sequence indicates that the mouse I C-peptide has a deletion of two amino acids compared with the mouse II C-peptide. We used an S1 nuclease assay to confirm the presence of the deletion and to measure the ratio of transcripts from gene I to transcripts from gene II. The mouse preproinsulin I gene, like the rat gene I, is missing the second intervening sequence that normally interrupts the C-peptide region in other insulin genes. Comparison of the 5' flanking sequences of the mouse and rat genes II indicates that they are homologous for at least 1000 base pairs. The preproinsulin I genes also share homology in their 5' flanking DNAs; however, their homology to the preproinsulin II genes extends for only about 500 base pairs.

Amino Acid Sequence↗
PubMed1982

Construction of cloned libraries from RNA of human fetal tissues.

We have constructed libraries of recombinant DNA plasmids containing sequences complementary to polyadenylated RNA from a variety of human midtrimester fetal tissues. The bacterial colonies containing these plasmids have been grown and replicated on nitrocellulose filters in a manner that facilitates permanent storage, rapid screening, and transportability to other laboratories. We screened a portion of the library for the presence of repetitive sequences and found that approximately 20% of the clones contain repetitive sequences. We have also shown that some clones contain nonrepetitive sequences. Pools of recombinant cDNA-containing plasmids devoid of repetitive sequences have been constructed to permit the chromosomal localization of a variety of actively transcribed sequences. The construction of such large, tissue-specific clone banks should facilitate the direct isolation, mapping, and characterization of normal and abnormal human genes.

Cloning, Molecular↗
PubMed1980

Chorion cDNA clones of D. melanogaster and their use in studies of sequence homology and chromosomal location of chorion genes.

Clones corresponding to two distinct A1 and A2 chorion genes have been isolated from a cDNA library in Drosophila melanogaster and characterized by hybrid-selected translation and blotting-hybridization analysis. These sequences detectably cross hybridize, thus indicating that at least some chorion genes in the fruit fly are homologous. According to in situ hybridization results, the A1 and A2 genes are not linked (mapping in regions 66D 10-12 and 54C-D of the third and second chromosomes, respectively). In conjunction with other evidence, these results suggest that in Drosophila, clustering of chorion genes may be limited to genes which are expressed in parallel during development.

Animals↗
PubMed1978

A bacterial clone synthesizing proinsulin.

We have cloned double-stranded cDNA copies of a rat preproinsulin messenger RNA in Escherichia coli chi1776, using the unique Pst endonuclease site of plasmid pBR322 that lies in the region encoding amino acids 181-182 of penicillinase. This site was reconstructed by inserting the cDNA with an oligo(dG)-oligo(dC) joining procedure. One of the clones expresses a fused protein bearing both insulin and penicillinase antigenic determinants. The DNA sequence of this plasmid shows that the insulin region is read in phase; a stretch of six glycine residues connects the alanine at position 182 of penicillinase to the fourth amino acid, glutamine, of rat proinsulin.

Base Sequence↗
PubMed1975

Initiation sites for translation of sindbis virus 42S and 26S messenger RNAs.

Sindbis virus 26S RNA is the principal species of virus-specific RNA found in the infected cell; it is derived from a one third segment of virion 42S RNA. When translated in cell-free extracts from mouse ascites cells or rabbit reticulocytes, 26S RNA directed the synthesis primarily of the 33,000 dalton virus capsid protein, and the protein products were in the form of free peptides rather than peptidyl-tRNA. In contrast, the polypeptides synthesized in either extract in response to Sindbis virus 42S RNA were heterogeneous, ranging in molecular weight from 33,000 to 190,000, and were largely in the form of peptidyl-tRNA. The number of independent initiation sites on the 26S and 42S RNAs was determined by analyzing a tryptic digest of reaction products labeled with yeast N-formyl-35S-methionyl-tRNAFmet. The 26S RNA appeared to contain a single initiation site, and this site could also be found in varying amounts in different preparations of 42S RNA. However, a second initiation site, distinct from that of 26S RNA, was the major site in 42S virion RNA. These results suggest that 42S virion RNA contains two potential sites for initiation of protein synthesis. Only one of these may be active, however, and it is postulated that the second site functions primarily, if not exclusively, in the subgenomic 26S RNA species. In this regard, Sindbis virus 42S RNA may represent a novel form of a eucaryotic messenger RNA.

Binding Sites↗
PubMed1975

Studies on the function of polyadenylic acid on poliovirus RNA.

RNAase H was used to remove selectively over 83% of 3' terminal polyadenylic acid [poly(A)] from poliovirus RNA. In a single cycle infection with this poly(A)-deficient RNA, no evidence for multiplication of the RNA was found. To elucidate further the function of poliovirus poly (A), the translational capacity of the RNAase H-treated and untreated RNA was studied in a cell-free protein synthesizing system prepared from HeLa cells. Removal of most of the poly(A) did not affect the pattern or amount of polypeptides synthesized. We conclude that the poly(A) on poliovirus RNA is probably necessary to the replication of the molecule.

Cell-Free System↗
PubMed1975

Complete translation of poliovirus RNA in a eukaryotic cell-free system.

Poliovirus RNA stimulates imcorporation of 35S from both [35S]methionine and formyl-[35S]methionyl-tRNAfMet in cell-free systems derived from HeLa cells or from poliovirus-infected HeLa cells. The largest product formed under the direction of the viral RNA is the same size as the polyprotein thought to represent translation of the entire RNA. Synthesis of this polyprotein and other large products was stimulated greatly by increasing the salt concentration during the reaction from the optimum for initiation (90 mM) to the optimum for elongation (155 mM). Only one initiation peptide could be identified, and a tryptic digest of the product contained mainly peptides that cochromatographed with peptides from authentic viral proteins. The RNA from a deletion mutant of poliovirus initiated protein synthesis at the same site used by standard RNA and programmed synthesis of an appropriately deleted set of polypeptides. The results strongly support the model of translation of poliovirus RNA from a single initiation site into a continuous polyprotein that is cleaved to form the functional proteins. It is suggested that uninfected HeLa cell extracts can carry out the cleavages of nascent polyprotein.

Cell-Free System↗
PubMed1972

Translation of reovirus messenger RNAs synthetesized in vitro into reovirus polypeptides by several mammalian cell-free extracts.

Single-stranded reovirus RNA, synthesized in vitro by reovirus cores, functioned as messenger RNA in cell-free extracts prepared from several mammalian cells: Krebs II mouse ascites cells, mouse L cells, Chinese hamster ovary cells, HeLa cells, and rabbit reticulocytes. As shown by acrylamide gel electrophoresis, all eight polypeptides known to be specified by reovirus were synthesized in the reticulocyte system. In the other extracts, from 5 to 7 complete virus proteins were made.

Animals↗
PubMedDate not supplied

Biological activity of rat proinsulin synthesized by Escherichia coli.

Fused proteins possessing insulin antigenic determinants are synthesized and secreted by several strains of Escherichia coli K-12 bearing plasmids containing cDNA copies of rat proinsulin mRNA. Small amounts of the secreted, fused protein from E. coli strains chi 1776 and HB101 bearing plasmid p147 were partially purified and assayed for biological activity by determining stimulation of [1-14C]glucose oxidation to 14CO2 in the rat epididymal fat pad assay. Guinea pig anti-insulin serum (GPAIS) was used to suppress glucose oxidation stimulated by insulin. After mild treatment with trypsin, fused protein stimulated 14CO2 production, and over 90% of this activity was suppressible by GPAIS. Untrypsinized fused protein demonstrated no GPAIS-suppressible 14CO2 production. Larger amounts of fused protein were obtained by transformation of strain PR13 of E. coli in which the yield of eukaryotic proteins is increased approx. 50-fold. A single intravenous injection of trypsinized fused protein from E. coli PR13 containing plasmid p287.47 resulted in a rapid decline in the plasma glucose levels of fasted mice, and the hypoglycemic effect persisted for at least 120 min.

Adipose Tissue↗