Subject(s)
Antigens, Viral/biosynthesis , Hemagglutinins, Viral/biosynthesis , Influenza A virus/genetics , Amino Acid Sequence , Antigens, Viral/genetics , Antigens, Viral/immunology , Base Sequence , Escherichia coli/genetics , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/immunology , History, 20th Century , Influenza A virus/immunology , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
Commercial activities frequently take place against a background of the ownership of intellectual property, for example patents or know-how. This is particularly true of human health care companies and companies operating in the field of biotechnology, where the high cost and long time frames of research and development demand a degree of exclusivity in order to achieve an acceptable return on the investment. It follows that any company with an interest in using biotechnology to generate human health care products will have a strong interest in the ownership of property, much of which will have its origins in knowledge of the human genome. Claims to the ownership of portions of the human genome have been made public in recent times and these have been treated with everything from disgust to derision. However, a more considered analysis of this question will be found to turn on the relationship between discovery and invention in this field. This paper examines this question in relation to current commercial activities where information from the human genome is being used and attempts to draw some conclusions about what may be the impact of knowledge of the genome on commercial activities in the future.
Subject(s)
Biotechnology , Genome, Human , Patents as Topic , Ethics , HumansABSTRACT
DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.
Subject(s)
Chymosin/genetics , Cloning, Molecular , DNA , Enzyme Precursors/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , DNA Restriction Enzymes , Protein Biosynthesis , RNA, Messenger/genetics , Stomach/enzymologyABSTRACT
Experiments in which immobilised restriction fragments of genomic DNA were hybridised with a cloned human fibroblast interferon cDNA indicate that the homologous chromosomal genes exist in only one basic arrangement. This is in marked contrast to recent studies by Nagata et al. (1) showing that there are at least eight gene arrangements for human leukocyte interferon. Having isolated a chromosomal human fibroblast interferon gene from a gene bank, we conclude from nucleotide sequencing studies that there is a complete absence of introns within the RNA-coding region. In view of a similar observation recently made for a human leukocyte interferon gene (1), it would appear as if interferon genes in general are unlike the vast majority of eukaryote genes in this respect.
Subject(s)
Cloning, Molecular , Genes , Interferons/genetics , Base Sequence , Chromosomes, Human/metabolism , DNA Restriction Enzymes , DNA, Recombinant/metabolism , Fibroblasts/metabolism , Humans , Leukocytes/metabolism , Nucleic Acid Hybridization , Protein BiosynthesisABSTRACT
Two dodecadeoxynucleotides of defined sequence have been synthesised by phosphotriester methodology. They can be polymerised to give a double stranded DNA which codes, when read in the correct phase, for the repeating dipeptide poly(aspartyl-phenylalanine). This polymeric DNA has been cloned in E. coli K12 using as vector a plasmid having a controllable bacterial promoter upstream of the insertion site. Clones containing genes coding for up to 150 repeats of (aspartyl-phenylalanine) have been isolated and characterised. The polymeric inserts appear to be stable over many generations and are expressed in E. coli under the control of the bacterial promoter, to give a polymer of phenylalanine and aspartic acid which may be broken down enzymically to yield aspartyl-phenylalanine.
Subject(s)
DNA, Recombinant , Escherichia coli/metabolism , Genes , Peptide Biosynthesis , Peptides , Amino Acid Sequence , Cloning, Molecular , Plasmids , Protein Biosynthesis , Repetitive Sequences, Nucleic Acid , Transcription, GeneticABSTRACT
Using synthetic oligodeoxyribonucleotides to prime the transcription of interferon mRNA and cDNA, we recently determined the mRNA sequence coding for the 47 amino-terminal amino acids of mature human fibroblast interferon (1). From this sequence, we have now synthesised an oligodeoxyribonucleotide that is homologous with the mRNA sequence coding for amino acids 42-45 and used it as a primer to selectively transcribe an interferon cDNA template. The sequence of the newly synthesised DNA predicted the sequence of amino acids 48-109 in the interferon polypeptide. By repeating this process with one more primer, we have determined the complete amino acid sequence of mature human fibroblast interferon, a polypeptide of 166 amino acids.
Subject(s)
Interferons/biosynthesis , Oligodeoxyribonucleotides , Oligonucleotides , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase/metabolism , Transcription, Genetic , Amino Acid Sequence , Base Sequence , Fibroblasts/metabolism , Humans , Molecular Weight , Nucleic Acid HybridizationABSTRACT
From recently published data on the amino-terminal structures of human and mouse interferons, we have predicted and synthesised an oligonucleotide capable of priming specifically the reverse transcription of human fibroblast interferon mRNA present within a total mRNA population. From these transcripts we determined the sequence of the 5'-terminus of the mRNA and identified a putative pre-peptide signal sequence. This enabled us to predict the sequence of another primer capable of directing the synthesis of interferon double-stranded cDNA corresponding to the entire coding region of the mRNA. Further sequencing studies also enabled us to establish the identity of 47 consecutive amino acids beginning with the methionine residue at the amino-terminus of the mature protein.
Subject(s)
Fibroblasts/metabolism , Genes, MHC Class II , Interferons/genetics , Amino Acid Sequence , Humans , Oligodeoxyribonucleotides , Oligonucleotides , Poly T , RNA, Messenger/genetics , RNA-Directed DNA PolymeraseABSTRACT
A gene sequence for the fowl plague virus (FPV) haemagglutinin molecule has been inserted into a bacterial plasmid such that its transcription is under the control of a promoter derived from the tryptophan operon. Such plasmids direct the synthesis of a protein that reacts specifically with antisera to FPV haemagglutinin. Evidence is also presented that in some cases DNA inserted at the HindIII site of pBR322 is expressed.
Subject(s)
Antigens, Viral/genetics , DNA, Recombinant , Escherichia coli/genetics , Hemagglutinins, Viral/genetics , Influenza A virus/genetics , Epitopes , Genes , Influenza A virus/immunology , Operon , Plasmids , Transcription, Genetic , Tryptophan/geneticsABSTRACT
A synthetic fowl plague virus (FPV) haemagglutinin gene has been cloned in bacteria and the complete sequence of the RNA gene deduced. It is 1,742 nucleotides long and the mRNA codes for 56.3 amino acids in an uninterrupted sequence. The nature of some of the important domains in the haemagglutinin has been established, and their structure is discussed in relation to their function. Extensive amino acid sequence homologies exist between FPV and human influenza haemagglutinins.
Subject(s)
Genes, Viral , Genes , Hemagglutinins, Viral/genetics , Influenza A virus/genetics , Viral Proteins/genetics , Amino Acid Sequence , Codon , DNA Repair , DNA, Recombinant , RNA, Messenger/geneticsABSTRACT
The polyadenylation of Fowl Plague Viral RNA and of Influenza A/Victoria Viral RNA using E. coli poly (A) polymerase and the subsequent reverse transcription of the polyadenylated species is reported. We have shown that all 8 genome fragments are adenylated and that an average of 25--30 adenylic acid residues per molecule is sufficient for maximal transcription with reverse transcriptase. The cDNA product is 95% sensitive to Sl-nuclease and hybridisation analysis against viral RNA reveals it to be a faithful copy of the RNA. Amongst the transcription products are long, discrete copies of genes 1--8, the lengths of which are comparable with those of the vRNA determined by electrophoresis on formamide acrylamide gels. These single-stranded cDNAs have been further transcribed to form double-stranded products with hair-pin structures at one end. Analysis of this material on native acrylamide gels revealed some DNA bands corresponding to the predicted sizes for genes 4--8.
Subject(s)
Escherichia coli/enzymology , Nucleotidyltransferases/metabolism , Poly A/biosynthesis , Polynucleotide Adenylyltransferase/metabolism , RNA, Viral/metabolism , RNA-Directed DNA Polymerase/metabolism , DNA, Viral/biosynthesis , Influenza A virus , Kinetics , Molecular Weight , Nucleic Acid Hybridization , OrthomyxoviridaeSubject(s)
DNA , Genes , Ovalbumin/biosynthesis , RNA, Messenger/metabolism , Animals , Base Sequence , Chickens , DNA/metabolism , DNA Restriction Enzymes , Molecular Weight , Transcription, GeneticABSTRACT
Chicken DNA has been digested with restriction enzymes and the size distribution of the DNA fragments containing ovalbumin specific sequences has been examined after separation of the fragments on agarose gels and transfer to nitrocellulose sheets. Hybridisation with terminally 32P-labelled ovalbumin mRNA fragments or with RNA populations transcribed from the DNA of a hybrid plasmid containing ovalbumin sequences was used to locate the DNA fragments coding for ovalbumin. Digestion with enzymes which do not cut within the portion of the ovalbumin gene synthesised from ovalbumin messenger RNA in vitro has shown the presence of several defined fragments carrying ovalbumin specific sequences. Possible explanations of these observations are discussed.
Subject(s)
DNA/genetics , Genes , Ovalbumin/genetics , RNA, Messenger/genetics , Animals , Chickens/genetics , DNA Restriction Enzymes , Liver , Molecular Weight , Nucleic Acid Hybridization , Oviducts , PlasmidsABSTRACT
The presence in encephalomyocarditis (EMC) virus RNA of homonucleotide tracts 10 nucleotides or more in length has been investigated by testing the ability of homo-oligodeoxynucleotides to prime DNA synthesis in the reverse transcriptase from avian myeloblastosis virus. Neither (dC)10 nor (dA)10 promoted incorporation of [3H]deoxynucleotides into acid-insoluble material but (dG)10 and (dT)12-18 were effective primers and produced DNA products approximately 2000 nucleotides in length. We conclude that there are single-stranded oligo(rC) and oligo(rA) tracts in native EMC virus RNA at 37 degrees C. Kinetic analysis indicated that oligo(dT) priming is similar to priming on ovalbumin mRNA and that it gives rise to only one DNA product per template molecule. Oligo(dG) priming appears to be complicated by self-aggregation of the primer. Oligo(dT)-primed and oligo(dG)-primed DNA have both been separated on alkaline-sucrose gradients into two peaks of which only the 'heavier' will hybridise to EMC virus RNA. Competitive hybridisation experiments indicate that the 'heavy' oligo(dT)-primed and oligo(dG)-primed DNA fractions hybridise to overlapping sequences of EMC virus RNA and place the priming regions of EMC virus RNA approximately 500 nucleotides apart during reverse transcription.