Construction of chimeric plasmids containing histone H5 cDNA from hen erythrocyte. DNA sequence of a fragment derived from the 5' region of H5 mRNA.

We report that construction and characterization of chicken erythrocyte histone H5 cDNA recombinant plasmids. cDNA was synthesized from poly(A)+ polysomal RNA enriched in H5 mRNA and inserted into the PstI site of pBR322. Several clones containing H5 cDNA sequences were obtained and one of them (p541), expressing H5 antigenic determinants, was sequenced. The DNA insert of p541 contains 118 nucleotides from the 5' non-translated region of H5 mRNA and sequences coding for up to residue 46 of the N-terminus of the arginine (position 15) H5 variant. There is a strikingly high number of repeated sequences both in the leader and coding region; among these, the octanucleotide 5' GCG GCG GC 3' is found five times along the sequence. Although the H5 mRNA 5' leader is GC-rich (66%), there is an AT-rich region, about 16 nucleotides long, which shares strong homology with the leaders of sea urchin histone H1 mRNAs.     

Construction and analysis of recombinant DNAs containing a structural gene for rat prolactin.

Poly A-containing RNA enriched in prolactin-coding sequences was isolated from female rate pituitaries after induction with diethylstilbesterol. Double stranded cDNA was synthesized from this RNA and inserted into plasmid pBR322 at the Pst I site via the poly(dG):polyy(dC) tailing method. E. coli was transformed with this DNA and the recombinant plasmid in one of the transformants characterized in detail. About half of its 900 base pair cDNA insert was sequenced. The DNA sequence is consistent with most of the reported amino acid sequence of rat preprolactin. In addition, the recombinant plasmids in two of the other transformants appear to contain growth hormone coding sequences.     

Cloning of double stranded DNAs derived from polysomal mRNA of maize endosperm: isolation and characterisation of zein clones.

Starting from polysomal RNA of developing maize endosperm and applying the cloning procedure of integrating dsDNA into the Pst I site of plasmid pBR 322, clones containing sequences complementary to endosperm mRNAs were obtained. 25 per cent of these clones were identified as containing zein specific DNA sequences which hybridized either with the zein mRNA coding for the 22 000 Mr protein or with the zein mRNA coding for the 19 000 Mr protein. The zein-specific DNA inserts of the recombinant plasmids were further characterized by restriction enzyme analysis.     

Synthesis of part of a mouse immunoglobulin light chain in a bacterial clone

We have cloned double stranded cDNA sequences encoding a mouse immunoglobulin light chain (L-321) into the PstI site of the beta-lactamase gene of plasmid pBR322 by the oligo (dG)-oligo (dC) tailing procedure. Escherichia coli X1776 transformed by the recombinant plasmids were screened for the expression of L-321 antigenic determinants by a newly developed in situ radio-immunoassay. One out of seven transformants screened was found to synthesize an L-chain like protein. Each bacterial cell produces about 550 molecules of the L-chain sequence. Preferential segregation of the L-chain sequence to the periplasmic space suggest covalent attachment of the L-chain sequence to the N-terminal portion of beta-lactamase. Restriction mapping of the plasmid DNA isolated from the positive clone indicated the presence of a DNA sequence coding for the entire constant region and extending into the variable region for a length corresponding to about 40 amino acid residues. The orientation of the cloned cDNA with respect to the plasmid DNA is compatible with the formation of a fused beta-lactamase-L-321 peptide.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Expression in male and genomic organization of the gene(s) coding for a major protein secreted by the rat seminal vesicle epithelium.

Double strand cDNA copies of lls poly(A)+mRNA purified from adult rat seminal vesicles (RSV), have been cloned in E.coli C600 using the Pst I site of pBR322. Filter hybridization, nucleotide sequence analysis and positive hybridization translation were used to demonstrate that one of the recombinant plasmids obtained (pRSV25) contained a 260 bp long insert coding for a significant part of the precursor to the protein IV present in the RSV secretion. By using labelled pRSV25 DNA we have found that high levels of RSV IV mRNA were present only in the rat seminal vesicle epithelium. The amounts of RSV IV mRNA present in other tissues of the same organism were below the levels detectable by the methods used. In addition, other data reported here indicate that the RSV IV gene(s) is present in both sexes, probably with a different organization.     

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.     

Expression of poliovirus complementary DNA coding for viral antigenic determinants in Escherichia coli

DNA sequences coding for the immunogenic capsid protein VP1 and/or VP3 of poliovirus strain LSc-2ab (Sabin 1) were prepared by digesting the cloned complementary DNA with restriction endonuclease PstI. The DNA fragments were inserted into the unique PstI site of Escherichia coli plasmid vectors pBR322, pKT 280 and/or pKT 287 that lay in the region expressed under control of the penicillinase promoter system. In the expression vectors, poliovirus sequences were designed to be read in phase and therefore to be expressed as fusion proteins with the bacterial peptides. In addition, the Escherichia coli tryptophan operon promoter-operator system was inserted upstream of the penicillinase system to obtain stronger expression of the poliovirus sequences. Escherichia coli transformed with these plasmids appeared to produce the antigenic polypeptides, which were detected by immunoprecipitation with antibodies to capsid proteins VP1 and/or VP3 followed by SDS-polyacrylamide gel electrophoresis.     

Androgen regulation of prostatic binding protein messenger RNA studied by using cloned complementary DNA

The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.     

Albumin is encoded by 2 messenger RNAs in Xenopus laevis

A cDNA clone library was prepared from liver poly(A) RNA pf non-estrogenized Xenopus laevis. Albumin coding sequences were screened by hybridization to a cDNA prepared from poly(A) RNA enriched by sucrose density gradient centrifugation, and by a sensitive solid-phase radioimmunoassay to detect clones that contain templates for albumin antigenic determinants. Nine clones were obtained by this approach, and all but one have the cDNA inserted in phase with the beta-lactamase gene of pBR322. Mapping of these clones with restriction endonucleases yielded 2 distinct patterns, suggestive of heterogeneity in the coding sequences. This was confirmed by heteroduplex analyses of hybrids formed between clones representative of each of the 2 classes. Both classes of albumin cDNA clones were used to select mRNAs of the same size (2.3kb) that code for peptides that are indistinguishable by SDS gel electrophoresis. Examination of the organization of the albumin genes by blot hybridization of the cDNA clones to restriction fragments of Xenopus DNA failed to detect any differences at the genomic level. The considerable diversity of the albumin cDNAs is suggestive of a multiplicity of albumin genes, rather than differential processing of a common precursor RNA.     

The complete sequence of human preprocalcitonin

DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin-producing tumour, was inserted in the Pst site of pBR 322 by G-C tailing. The recombinant plasmids were used to transform Escherichia coli DP 50. Ampicillin-resistant clones were screened using a 32P-labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA. Positive clones were subsequently rescreened using a 32P poly(T) probe. Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro. An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced. This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5'-end, and 295 bp upstream from the poly(A) tail. The complete amino acid sequence of human preprocalcitonin could thus be deduced.     

Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

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.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage.

Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells.     

Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein

Aequorin is a bioluminescent protein which consists of a polypeptide chain (apoaequorin), coelenterate luciferin, and bound oxygen. Aequorin produces blue light upon binding Ca2+. We have isolated six recombinant pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic oligonucleotide probe was used to identify these cDNAs. An extract of an E. coli strain possessing the largest cDNA contained apoaequorin. This apoaequorin can be converted to aequorin in the presence of coelenterate luciferin, 2-mercaptoethanol, and O2. This cDNA is therefore apparently full-length.     

Isolation and characterization of a fragment of rat thyroglobulin gene

A rat genomic library was screened for thyroglobulin gene clones with recombinant plasmids containing rat thyroglobulin complementary DNA inserts. Two identical recombinant phages were found. A map of the inserted genomic sequence established by restriction and blotting experiments, and electronic microscopy revealed that this fraction of the gene was extensively split. Exons were ≤ 200 base pair long while the introns represented 93% of the insert. A fragment subcloned in plasmid pBR 322 was shown to contain repetitive sequences when used in Southern blot experiments with rat total genomic DNA.     

Transformation of rat fibroblasts by cloned defective polyoma DNA

Defective polyoma DNA molecules isolated from mouse cells infected with high-multiplicity-passaged virus were cloned in pBR322, and the recombinant plasmids were tested for their capacity to transform Fischer rat 3T3 cells in culture. Recombinants carrying an intact proximal portion of the early region, i.e., the region coding for both small and middle T antigens, were able to induce the transformed phenotype. A recombinant plasmid containing a defective polyoma genome with a deletion of about 300 base pairs in the region coding for the C-terminal segment of middle T antigen failed to transform.