Showing papers on "Complementary DNA published in 1979"
TL;DR: The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-β-lipotropin precursor mRNA indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion.
Abstract: The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.
1,689 citations
TL;DR: This ‘hybrid’ gene was expressed in Escherichia coli under the control of the lac promoter and a polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.
Abstract: DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This ‘hybrid’ gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.
674 citations
TL;DR: The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence.
Abstract: The nucleotide sequence of a DNA complementary to human growth hormone messenger RNA was cloned; it contains 29 nucleotides in its 5' untranslated region, the 651 nucleotides coding for the prehormone, and the entire 3' untranslated region (108 nucleotides). The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and, by comparison with the previously determined sequences of rat growth hormone and human chorionic somatomammotropin, strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence. The human growth hormone gene sequences have been linked in phase to a fragment of the trp D gene of Escherichia coli in a plasmid vehicle, and a fusion protein is synthesized at high level (approximately 3 percent of bacterial protein) under the control of the regulatory region of the trp operon. This fusion protein (70 percent of whose amino acids are coded for by the human growth hormone gene) reacts specifically with antibodies to human growth hormone and is stable in E. coli.
410 citations
TL;DR: Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size, and indicated that part of this difference is attributable to intervening sequences in the CAD gene.
407 citations
TL;DR: Rabbit β-globin complementary DNA has been inserted into SV40DNA in place of the gene coding for the virus' major capsid protein, VP1, and the recombinant genome, SVGT5-RaβG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the β- globin coding sequence.
Abstract: Rabbit beta-globin complementary DNA (cDNA) has been inserted into SV40 DNA in place of the gene coding for the virus' major capsid protein, VP1. The recombinant genome, SVGT5-RabetaG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the beta-globin coding sequence. Cells propagating SVGT5-RabetaG produce substantial quantities of rabbit beta-globin polypeptide.
365 citations
TL;DR: A 621-base pair fragment of the cDNA for the α-subunit of human chorionic gonadotropin has been isolated by cloning in a plasmid vector, and the complete nucleotide sequence determined.
Abstract: A 621-base pair fragment of the cDNA for the α-subunit of human chorionic gonadotropin has been isolated by cloning in a plasmid vector, and the complete nucleotide sequence determined. The entire coding region, including the 24-amino acid pre-sequence and most of the untranslated regions, are present in the fragment.
293 citations
TL;DR: Multiple nitrocellulose DNA filter replicas of plaques of in vitro generated recombinants of phage lambda and Saccharomyces cerevisiae have been screened by hybridization with 32P-labeled cDNA probes to allow the use of specific differences in total RNA populations as probes for gene isolation.
250 citations
TL;DR: The data suggest that evolution of oncogenic retroviruses has frequently involved a mechanism whereby incorporation and perhaps modification of different host genes provides each virus with the ability to induce its characteristic tumors.
Abstract: The avian carcinoma virus MC29 (MC29V) contains a sequence of approximately 1,500 nucleotides which may represent a gene responsible for tumorigenesis by MC29V. We present evidence that MC29V has acquired this nucleotide sequence from the DNA of its host. The host sequence which has been incorporated by MC29V is transcribed into RNA in uninfected chicken cells and thus probably encodes a cellular gene. We have prepared radioactive DNA complementary to the putative MC29V transforming gene (cDNA(mc) (29)) and have found that sequences homologous to cDNA(mc) (29) are present in the genomes of several uninfected vertebrate species. The DNA of chicken, the natural host for MC29V, contains at least 90% of the sequences represented by cDNA(mc) (29). DNAs from other animals show significant but decreasing amounts of complementarity to cDNA(mc) (29) in accordance with their evolutionary divergence from chickens; the thermal stabilities of duplexes formed between cDNA(mc) (29) and avian DNAs also reflect phylogenetic divergence. Sequences complementary to cDNA(mc) (29) are transcribed into approximately 10 copies per cell of polyadenylated RNA in uninfected chicken fibroblasts. Thus, the vertebrate homolog of cDNA(mc) (29) may be a gene which has been conserved throughout vertebrate evolution and which served as a progenitor for the putative transforming gene of MC29V. Recent experiments suggest that the putative transforming gene of avian erythroblastosis virus, like that of MC29V, may have arisen by incorporation of a host gene (Stehelin et al., personal communication). These findings for avian erythroblastosis virus and MC29V closely parallel previous results, suggesting a host origin for src (D. H. Spector, B. Baker, H. E. Varmus, and J. M. Bishop, Cell 13:381-386, 1978; D. H. Spector, K. Smith, T. Padgett, P. McCombe, D. Roulland-Dussoix, C. Moscovici, H. E. Varmus, and J. M. Bishop, Cell 13:371-379, 1978; D. H. Spector, H. E. Varmus, and J. M. Bishop, Proc. Natl. Acad. Sci. U.S.A. 75:4102-4106, 1978; D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature [London] 260:170-173, 1976), the gene responsible for tumorigenesis by avian sarcoma virus. Avian sarcoma virus, avian erythroblastosis virus, and MC29V, however, induce distinctly different spectra of tumors within their host. The putative transforming genes of these viruses share no detectable homology, although sequences homologous to all three types of putative transforming genes occur and are highly conserved in the genomes of several vertebrate species. These data suggest that evolution of oncogenic retroviruses has frequently involved a mechanism whereby incorporation and perhaps modification of different host genes provides each virus with the ability to induce its characteristic tumors.
249 citations
TL;DR: A DNA sequence preceding this 5 "capping" site in the rat insulin I gene, TATAAAGC, is homologous to corresponding regions in other eucaryotic genes that have been proposed as putative promoter sites for the initiation of transcription.
210 citations
TL;DR: It is suggested that vitellogenin is encoded by a small family of related genes in Xenopus, as well as two main groups of sequences which differ from each other in approximately 20% of their nucleotides.
189 citations
TL;DR: Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to λCβG1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian β-globin genes.
TL;DR: The data suggest the presence of a prinicipal 5' terminus of early lytic and transformed cell mRNA's at residues 5152 to 5154 and raise the possibility of additional5' termini at one or more locations in the 0.62 to 0.659 m.u. region of these mRNA's.
Abstract: We have studied the structure of polyadenylated virus-specific cytoplasmic mRNA's in mouse and human cells transformed by simian virus 40 and in monkey cells infected with simian virus 40 in the presence of cytosine arabinoside by means of reverse transcriptase-catalyzed complementary DNA synthesis and complementary DNA sequencing. Abundant mRNA species containing splices from residues 4490 to 4557 (0.533 to 0.546 map units [m.u.]) and 4490 to 4837 (0.533 to 0.600 m.u.) were identified in both transformed and infected cells. Two principal reverse transcriptase stops were observed at the 5' termini of these mRNA's, both occurring with approximately equal frequency. The most distal of these stops was localized at residues 5152 to 5154 (0.660 m.u.), and the second was at residues 5147 to 5148 (0.659 m.u.). Several additional minor stops, between approximately 0.62 and 0.65 m.u., were also found on complementary DNA copied from transformed cell mRNA; in contrast, only one additional stop was present on complementary DNA copied from early lytic mRNA. These data suggest the presence of a prinicipal 5' terminus of early lytic and transformed cell mRNA's at residues 5152 to 5154 and raise the possibility of additional 5' termini at one or more locations in the 0.62 to 0.659 m.u. region of these mRNA's. Transformed cell mRNA was also found to contain a single 3' terminus at positions 2504 and 2505 (0.153 m.u.); termini lying beyond this site were not detected.
TL;DR: The cloning of a cDNA prepared from human insulin mRNA and an analysis of the nucleotide sequence of the cloned molecule including the region coding for the prepeptide and portions of the 5′- and the 3′-untranslated regions of the molecule are reported.
Abstract: Insulin consists of two polypeptide chains, A (21 amino acids) and B (30 amino acids), linked by disulphide bonds. Both chains are derived from one precursor, proinsulin, which includes a connecting peptide (C) between the A and B chains, and which is excised before the secretion of insulin from the pancreatic B cells1. The observation that the C-peptide varies between species, in contrast to the highly conserved A and B sequences1–3, is consistent with the theory that it serves a purely structural function in insulin synthesis. During in vitro translation of insulin mRNA, a larger peptide containing about 25 additional residues at the N-terminal end (preproinsulin) is the primary product4–7. The prepeptide is cleaved to leave proinsulin during transport into the endoplasmic reticulum and is thought to direct this process specifically8. Using automated amino acid sequence analysis, the partial amino acid sequences of the prepeptide regions of bovine, rat, sea raven and anglerfish preproinsulin have been established4,5,7. The nucleotide sequences of the cloned cDNA and gene coding for rat insulin I have confirmed the amino acid sequence of rat proinsulin I, and have also predicted the sequence of the prepeptide9–11. Like the prepeptides of other secreted proteins, this prepeptide has a prominent hydrophobic region12. We report here the cloning of a cDNA prepared from human insulin mRNA and an analysis of the nucleotide sequence of the cloned molecule including the region coding for the prepeptide and portions of the 5′- and the 3′-untranslated regions of the molecule. We also compare the structure of the human molecule with the previously reported rat mRNA9–11.
TL;DR: It is shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.
TL;DR: The chapter describes the procedures used to isolate and clone specific hormone cDNAs from an impure mRNA population using the restriction endonuclease cleavage of double-stranded cDNA transcribed from a complex mixture of mRNA.
Abstract: Publisher Summary This chapter discusses the cloning of hormone genes from a mixture of complementary deoxyribonucleic acid (cDNA) molecules. The molecular cloning of cDNA synthesized from a purified messenger ribonucleic acid (mRNA) is a well-established method for obtaining purified DNA for sequence analysis and use as a hybridization probe. Only a limited number of purified mRNAs are currently available that include those from specialized cells producing predominantly a single protein, such as globin, immunoglubin, or ovalbumin. The chapter describes the procedures used to isolate and clone specific hormone cDNAs from an impure mRNA population. The method employs the restriction endonuclease cleavage of double-stranded cDNA transcribed from a complex mixture of mRNA. The method does not require any extensive purification of RNA but instead makes use of the transcription of RNA into cDNA, the sequence-specific fragmentation of this cDNA, with one or two restriction endonucleases, and the fractionation of the cDNA restriction fragments on the basis of their lengths. The use of restriction endonucleases eliminates size heterogeneity and produces homogeneous-length DNA fragments from any cDNA species that contains at least two restriction sites. From the initially heterogeneous population of cDNA transcripts, uniform-sized fragments of desired sequence are produced. The chapter discusses a complete scheme for cDNA cloning.
TL;DR: Comparison of the nucleotide sequence of the genomic gene and cDNA containing the sequence complementary to fibroin mRNA has confirmed the existence of an intervening sequence 970 bases long, and pinpointed the 5′ coding-intervening junction at +64−66 and the 3′ intervening-coding junction at -1034−1036.
TL;DR: In this paper, a deoxyoligonucleotide probe was used to detect gastrin mRNA in poly(A)-enriched RNA preparations from hog antrum, and the nucleotide sequence of the oligonucleotide was deduced from the unique amino acid sequence Trp-Metglu-Glu of gastrin.
Abstract: We have used a specific deoxyoligonucleotide probe to detect gastrin mRNA in poly(A)-enriched RNA preparations from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. When used with hog antral RNA, the dodecanucleotide is an effective primer for the synthesis of gastrin-specific cDNA as judged by nucleotide sequence analysis of cDNA isolated by polyacrylamide gel electrophoresis. We have determined an 81-nucleotide sequence corresponding to the region of the gastrin mRNA that codes for the known amino acid sequence of the G34 progastrin intermediate species, and we have demonstrated the presence of two consecutive basic residues preceding the G34 sequence in the prohormone. Hybridization of gastrin cDNA or synthetic dodecanucleotide to hog antral RNA separated by gel electrophoresis on agarose gels in the presence of methylmercuric hydroxide indicates that the mRNA coding for gastrin is about 620 nucleotides long. These results suggest that the gastrin precursor peptide contains 110-140 amino acids. This method should be of general application for detection and characterization of mRNAs corresponding to proteins of known amino acid sequence.
TL;DR: The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.
TL;DR: A study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5 concludes that a 14 kb fragment, G3, contains three copies of the gene coding forThe 70K protein.
TL;DR: The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.
Abstract: The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.
TL;DR: The “dideoxy” sequencing method has been used to obtain 121 nucleotides of sequence of cDNA transcribed from the 3′ end of the RNA segment which contains the coding information for the hemagglutinin of influenza virus A/RI/5−/57 (H2N2).
TL;DR: A cDNA fragment synthesized from mouse mRNA that codes for the precursor polypeptide containing corticotropin, beta-lipotropic peptides, and several other peptides has been cloned in bacteria and it appears that there is extensive homology of mouse beta-LPH with human and porcine beta- LPH.
Abstract: A cDNA fragment synthesized from mouse mRNA (ACTH/LPH mRNA) that codes for the precursor polypeptide containing corticotropin (ACTH), beta-lipotropin (LPH), and several other peptides has been cloned in bacteria. The mRNA was enriched for ACTH/LPH mRNA translational activity (to about 75%) prior to cDNA synthesis. It appears to contain about 1200 bases, of which approximately 450 bases are not translated. The cloned DNA fragment is complementary to the region of the mRNA coding for the protein fragment beta-LPH-(44--90); this contains all of the amino acids of [Met]-enkephalin (residues 61--65 of beta-LPH), most of the amino acids of beta-melanocyte-stimulating hormone, and all but the carboxy-terminal amino acid of beta-endorphin. Based on assignment of the amino acid sequence of mouse beta-LPH from the nucelic acid sequence, it appears that there is extensive homology of mouse beta-LPH with human and porcine beta-LPH. The data also establish the linkage between beta-melanocyte-stimulating hormone and beta-endorphin as a Lys-Arg sequence. It is hoped that this cloned DNA can be used as a probe to study the expression and structure of the ACTH/LPH gene.
TL;DR: Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence, and partial DNA sequence analysis of the h GH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.
Abstract: A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2--3 kb was ligated to lambda gt WES.lambda B DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment.
TL;DR: An insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II is Generation using a synthetic deoxydecanucleotide.
Abstract: We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.
TL;DR: The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes.
TL;DR: The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined and show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin.
Abstract: The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin. The region 5' to the AUG initiating codon is greater than 90% A+T residues in all of the genes.
TL;DR: In this paper, molecular cloning and nucleotide sequence analysis of adult chicken beta globin mRNA was reported, and DNA sequences derived from in vitro transcrption of Globin mRNA were purified and amplified as recombinant DNA using the plasmid pBR322.
Abstract: The molecular cloning and nucleotide sequence analysis of adult chicken beta globin mRNA is reported. DNA sequences derived from in vitro transcrption of globin mRNA were purified and amplified as recombinant DNA using the plasmid pBR322. Sequence analysis of several clones coding for beta globin strongly suggests that transcription errors may be generated near the 5' end of transcripts in vitro by reverse transcription. The complete sequence of the longest beta globin insert containing 51 bases of the 5' untranslated region as well as the complete coding and 3' untranslated regions has been determined.
TL;DR: This work determined the orientation of 4 immediate early (IE) mRNA's on the herpes simplex virus type 1 genome by mapping cDNA's complementary to the 3'-termini of messages and proposed a model which allows their synthesis from a circular template using a single virus promoter region.
Abstract: We have determined the orientation of 4 immediate early (IE) mRNA's on the herpes simplex virus type 1 genome by mapping cDNA's complementary to the 3'-termini of messages. These IE mRNA's are transcribed by a pre-existing cell RNA polymerase, and we propose a model which allows their synthesis from a circular template using a single virus promoter region. The promoter region, which is located in the two repetitive DNA regions which flank the short unique region of the virus genome, may serve to initiate bidirectional transcription of these IE mRNA's.
TL;DR: Zein messenger RNAs from maize endosperm were purified by successive oligo(dT)-cellulose chromatography and sucrose gradient centrifugation and shown to have a base composition characteristic of a poly(A)-containing RNA and to be transcribed by reverse transcriptase into complementary DNA.
Abstract: Zein messenger RNAs from maize endosperm were purified by successive oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis under denaturing conditions revealed the presence of two size classes of zein messenger RNAs of Mr 3.5 x 10(5) and 4.10 x 10(5). The mRNA was shown to synthesize the major zein polypeptides, to have a base composition characteristic of a poly(A)-containing RNA and to be transcribed by reverse transcriptase into complementary DNA. The r0t1/2 of the hybridization curve of cDNA hybridized to an excess of mRNA was shown to be 7 x 10(-2) M . s indicating that about 15 non-cross-hybridizing sequences are present in the zein mRNA preparations. The kinetics of cDNA annealing with an excess of maize DNA from 2 n cells suggest a ten-times reiteration of each mRNA sequence. This result is confirmed from saturation experiments, where in cDNA excess to DNA, the number of zein genes per haploid maize genome was estimated as about 120 copies. Similar experiments carried out on DNA from normal and mutant endosperms (3n cells) indicate the absence of large amplifications or deletions of zein genes in the tissue devoted to zein synthesis.
TL;DR: Direct proof of a change in mRNA concentration during D. discoideum development for individual high and medium abundance mRNA species is found and it is found that only a small fraction show such a change.