Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.     

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.     

Synthesis and cloning of DNA complementary to mRNA of the bovine mammary gland

Poly(A+)mRNA from bovine mammary glands was used to synthesize double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst1 site by means of oligo(dG)-oligo(dC) tailing. After transfection of Escherichia coli JC5183, recombinant plasmid library containing 5400 clones was screened by serial rounds of colony hybridization in situ to total [23P] poly(A+)mRNA and electrophoretically homogenious [32P]16SmRNA of mammary glands. Then hybrid selection of mRNA and subsequent in vitro translation of selected mRNAs were performed. In this manner, recombinant clones coding for alpha S1- beta-, kappa-casein were identified. cDNA clones range in size from 35% for beta-casein, 65% for alpha S1-casein to about 95% for kappa-casein, in comparison with their respective mRNAs.     

Cloning of mouse carbonic anhydrase mRNA and its induction in mouse erythroleukemic cells

Mouse carbonic anhydrase mRNA was detected in poly(A+) RNA of anemic spleens sedimenting as a RNA species at 14 S. Subsequently, poly(A+) RNA (12-16 S) was used as a template for the synthesis of double-stranded cDNA, which was inserted into the PstI site of pBR322 by oligo-dG:dC tailing. A recombinant plasmid containing carbonic anhydrase cDNA was identified by a positive hybridization selection assay and by partial DNA sequencing. Predicted amino acid sequences showed homology with the known sequences of rabbit and human carbonic anhydrase I and II. The clone contained sequences for most of the coding region and 600-700 base pairs at the 3' noncoding region of the mRNA. Hybridization analysis of poly(A+) RNA from uninduced and induced mouse erythroleukemic cells labeled for short and long time periods indicated that induction results in an increase of carbonic anhydrase mRNA in newly synthesized RNA.     

Molecular cloning of cDNA for rat glycine methyltransferase

Using a highly purified preparation of glycine methyltransferase mRNA, double-stranded cDNA was synthesized and inserted into the PstI site of pBR322. The resulting recombinant DNA was used to transform E. coli X 1776 by conventional methods. Among tetracycline-resistant transformants, a number of colonies were found to contain cDNA sequence for glycine methyltransferase as examined by hybrid-selected translation. A restriction endonuclease cleavage map was constructed covering about 720 base pairs. With the cDNA as the probe, the content of the glycine methyltransferase mRNA was quantitated in various rat tissues and was found to be proportional to the specific enzyme activity.     

Cloning and sequencing of cDNA for mouse liver metallothionein-I

Metallothionein mRNA was purified from liver of mice injected with cadmium. The corresponding double-stranded cDNA was prepared and inserted into the PstI site of plasmid pBR322. The resulting recombinant DNA was used to transform the RR1 strain of $\text{Escherichia}\text{coli}$. Clones resistant to tetracycline and sensitive to ampicillin were screened for the presence of metallothionein-specific restriction fragments in their plasmids. One plasmid, called M135, contains a cDNA insert covering the entire length of the mRNA for mouse liver metallothionein-I, except for the first 18 bases at the 5′ end.     

A cloning vehicle suitable for strand separation

A new plasmid has been constructed which contains a poly(A) : poly(dT) duplex segment of length approx. 100 base pairs (bp) inserted into the PvuII site of pBR322. This plasmid, pKH47, has all the other restriction sites of pBR322 available for insertion of foreign DNA, and has the same drug resistance genes as does the parental plasmid. The complementary strands of the linearized denatured plasmid DNA can be separated rapidly an efficiently by affinity chromatography with oligo(dA)- and oligo(dT)-cellulose columns in series. More than 90% of the input DNA is recovered as separated strands which can be annealed to form full length double-stranded molecules. One of the applications of the plasmid is to prepare separated complementary strands for sequencing by the chain-terminator technique using DNA primers. This application is illustrated by a sequencing example for a Drosophila DNA insert carrying a tRNA gene.     

Bleomycin-A2 complexes with poly(dA--dT): a proton nuclear magnetic resonance study of the nonexchangeable hydrogens

The mRNA coding for uteroglobin, a progesterone-induced uterine protein, has been partially purified from 4-day pregnant rabbit uterus. Double-stranded DNA synthesized from the partially purified mRNA preparation was inserted into the Pst I site of pBR 322. Bacterial transformants containing uteroglobin DNA sequences were identified by their ability to enrich for uteroglobin mRNA on hybridization with total uterine poly A-RNA. The identity of one recombinant was confirmed unambiguously by matching its nucleotide sequence with the amino acid sequence of the uteroglobin polypeptide.     

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.     

The influenza virus haemagglutinin gene: cloning and characterisation of a double-stranded DNA copy.

A protocol has been developed for the synthesis of a double-stranded DNA (dsDNA) copy of the influenza virus RNA genome segment which codes for the major surface antigen, haemagglutinin (HA). This dsDNA copy was inserted, after digestion with S1 nuclease and poly (dC) tailing with terminal transferase, into poly(dG)-tailed, PstI-cut, pBR322 DNA, and used to transform E. coli RR1. Tetracycline-resistant bacterial colonies were screened for the presence of plasmid containing the copied HA gene by testing their ability to hybridise to a specific, 32P-labelled, single-stranded DNA probe. Four cloned hybrid plasmids, containing DNA complementary to the HA gene of the influenza strain 29C (a laboratory derivative of influenza A/NT/60/68 (1)) were analysed by restriction enzyme mapping. Each contained a dsDNA insert equivalent to a full length copy of the HA gene. The nucleotide sequence of a selected restriction fragment from the DNA inserted in one of these cloned plasmids (C89) was determined. The amino acid sequence deduced from these data agreed with the amino acid sequence determined for the corresponding region of HA from the influenza strain A/Mem/102/72, another member of the Hong Kong subtype, identifying the inserted dsDNA of C89 as an authentic copy of the influenza HA gene.