Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome

Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.     

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.     

Molecular cloning of unintegrated visna viral DNA and characterization of frequent deletions in the 3' terminus

Visna viral DNA, like other retroviral DNA, exists in two circular forms in infected cells. The larger probably contains two copies of the LTR, the smaller, one copy. Recombinant DNA techniques were used to clone unintegrated circular visna viral DNA in the lambda WES . lambda B vector. Circular visna viral DNA was digested with the restriction enzyme SstI, which yields a 9.2-kb viral DNA fragment containing 90% of the viral genome colinear with the restriction map of linear viral DNA. This fragment extends from a site about 900 bp from the left (5') end of the viral DNA molecule, through the 3' region, including U3 and R sequences at its right (3') end. The recombinant clones isolated contain visna viral DNA inserts which range in size from 3.1 kb to 9.2 kb. All the clones contain the 5' region intact, but most had sustained deletions of varying lengths in the 3' terminal region of the cloned fragment.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

New versatile cloning and sequencing vectors based on bacteriophage M13

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Restriction map of Chinese hamster mitochondrial DNA containing replication coordinates: comparison with Syrian hamster mitochondrial genome

A precise physical map, containing the structurally and operationally defined D-loop origin, terminal region, and direction of heavy-strand replication, has been constructed for mitochondrial DNA (mtDNA) from ovary (CHO-KI) and lung cells of Chinese hamster (Cricetulus griseus 2 N = 22), and compared with our previously established genome coordinates for mtDNA from Syrian hamster ( Mesocricetus auratus 2 N = 44). All four HpaI sites in Cricetulus are conserved in Mesocricetus (8 sites). Extensive variation exists for hexanucleotides cleaved by EcoRI HindIII PstI. KpnI and BamHI. Sequence divergence between Chinese and Syrian hamster mtDNAs, as reflected from analysis of the mapped recognition sites for these six endonucleases, is estimated as 5–9% base substitutions. mtDNAs from both hamster and several other mammalian species contain a commonly conserved HpaI site in the region of light strand initiation.     

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.