A bacterial clone synthesizing proinsulin. 1978.
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Amino Acid Sequence↗
Biomedical subjects
A Efstratiadis
Publications and source records attributed to A Efstratiadis.
Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure.
A dot hybridization method is presented for rapidly determining the relative concentrations of nucleic acids in a mixture, as well as the extent of sequence homology between related RNA or DNA species.
Base Sequence↗
The structure and transcription of four linked rabbit beta-like globin genes.
Rabbit chromosomal DNA contains a cluster of four linked beta-like globin genes arranged in the orientation 5'-beta 4-(8kb)-beta 3-(5 kb)-beta 2-(7-kb)-beta 1-3'. Determination of the nucleotide sequence of gene beta 1 confirms that this gene corresponds to the second type of two common co-dominant alleles encoding the adult beta-globin chain. With the exception of two nucleotide substitutions in the large intervening sequence (intron), the intron and flanking sequences are identical with the nucleotide sequence of the first type determined by Weissmann et al. (1979). A 14S polyadenylated transcript containing large intron sequences (possibly a mRNA precursor) is detected in the bone marrow cells of anemic rabbits. Gene beta 2 has limited sequence homology to adult and embryonic beta-globin probes and lacks a detectable mRNA transcript in the erythropoietic tissues examined. It contains at least one intervening sequence analogous to the large intron in gene beta 1. Genes beta 3 and beta 4 both contain an intron of 0.8 kb. Partial DNA sequence analysis indicates that the large intron in beta 4 is located between codons for amino acids lysine and leucine in an analogous position to that of the large intron in beta 1. In addition, a second smaller intron interrupts the 5' coding sequences of gene beta 4. Both genes beta 3 and beta 4 are transcribed in embryonic globin-producing cells. Their DNA sequence homology is limited, however, to a segment of approximately 0.2 kb located on the 5' side of the large intron.
Animals↗
Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families.
A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. 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.
Animals↗
The 3' noncoding region of beta-globin mRNA is not essential for in vitro translation.
Rabbit beta globin DNA sequence, excised from plasmid pbetaG1, directs in vitro synthesis of beta-globin in a transcription-translation cell-free system, even after specific elimination of the entire 3'-noncoding region. A DNA restriction fragment carrying this 3' noncoding region and hybridized to globin mRNA cannot arrest the cell-free translation of beta-globin mRNA.
Animals↗
The isolation of structural genes from libraries of eucaryotic DNA.
We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.
Base Sequence↗
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↗
Insertion of synthetic copies of human globin genes into bacterial plasmids.
Double stranded human globin cDNA was synthesized by use of viral reverse transcriptase from globin mRNA of cord blood of premature infants requiring exchange transfusions. The cDNA was introduced into plasmids and the recombinant DNA plasmids used to transform E. coli X1776. A number of transformants were obtained. Plasmid DNA from selected colonies was isolated and characterized for the type of globin cDNA it contained by three types of procedures: 1) hybridization to previously characterized 3H-labeled alpha,beta and gamma cDNA; 2) analysis of the size and nature of fragments produced by digestion of the plasma DNA by different restriction endonucleases; and 3) by rapid DNA sequence analysis of selected DNA fragments produced by restriction endonuclease digestion. Analysis by these techniques of plasmid DNA from different colonies has definitively identified the presence of human alpha, beta or gamma cDNA sequences in different plasmids.
Base Sequence↗
Studies on the structure of genes expressed during development.
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Age Factors↗
Amplification and characterization of eukaryotic structural genes.
An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.
Animals↗
The eggshell of insects: differentiation-specific proteins and the control of their synthesis and accumulation during development.
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Animals↗
The primary structure of rabbit beta-globin mRNA as determined from cloned DNA.
The rabbit beta-globin DNA insertion of the hybrid plasmid PbetaG1 (Maniatis et al., 1976) was sequenced by the method of Maxam and Gilbert (1977). A sequence of 576 nucleotides was determined and verified by pyrimidine tract analysis of double-stranded DNA, synthesized in vitro starting from beta-globin mRNA. The derived sequence is in complete agreement with previously reported partial mRNA sequencing data and with the predictions from the primary structure of the protein. Moreover, the globin DNA insertion is missing only 13 nucleotides corresponding to the 5' terminal sequence of the mRNA. The rabbit beta-globin mRNA consists of a coding region of 438 nucleotides, flanked by a 5' noncoding region of 56 nucleotides (including the initiation codon AUG but not the 7-methyl-guanine of the "cap structure") and by a 3' noncoding region of 95 nucleotides (including a UGA termination codon). The features of the mRNA sequence are discussed with specific attention to the selective use of particular codons, the probable existence extensively base-paired segments at the 5' terminal region and the ribosome binding site. The faithful representation of beta-globin mRNA in the PbetaG1 DNA insertion establishes the validity of used cloned DNA, initially derived from double-stranded DNA transcripts of mRNA, for studying the structure of eucaryotic genes.
Animals↗
Molecular evolution of human and rabbit beta-globin mRNAs.
The primary structures of human and rabbit beta-globin mRNAs are compared. Using as a standard the extent of nucleotide substitutions inferred from the hypervariable amino acid residues of fibrinopeptides A and B, which are thought to change largely by neutral evolution, we show that not all silent mutations in globin mRNA are neutral. The divergence of the sequences is limited in part by the selective usage of synonymous codons. The divergent nucleotides tend to be distributed nonrandomly: in the coding region silent substitutions are most rare in segments that are also deficient in substitutions leading to replacements.
Animals↗
End labeling of enzymatically decapped mRNA.
A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.
Adenosine Triphosphatases↗
Synthesis of RNA complementary of rabbit globin mRNA by Qbeta replicase.
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Animals↗
Amplification and characterization of a beta-globin gene synthesized in vitro.
Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.
Base Sequence↗
Enzymatic in vitro synthesis of globin genes.
Full-length, single-stranded rabbit globin cDNA, synthesized by AMV reverse transcriptase, apparently contains a small double-stranded sequence (hairpin) at the 3' terminus. This cDNA can serve as template-primer for E. coli DNA polymerase I, which synthesizes a strand complementary to the cDNA and covalently bound to it. The loop connecting the two strands can be cut by S1 nuclease. Reassociation, hybridization, and restriction endonuclease studies, as well as electrophoretic analyses, indicate that the sequential actions of reverse transcriptase, DNA polymerase 1, and S1 nuclease generate full-length, double-stranded synthetic globin genes.
Animals↗
DNA sequence organization in the lepidopteran Antheraea pernyi.
A large fraction of the genome of the lepidopteran Antheraea pernyi consists of interspersed single-copy sequences and repetitive DNA sequences of 300 nucleotide average length. Most of the single-copy sequences are about 800 nucleotides long and a minority are considerably longer. Thus, the organization of the DNA sequences of this insect is similar to that of most higher organisms and different from that of Drosophila.
Animals↗