Structure of recombinant plasmids containing synthetic human foetal globin gene sequences

Summary In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid or substitution loops due to insertion of globin DNA sequences combined with deletions of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.     

Cloning of chicken globin cDNA in bacterial plasmids

Chicken globin cDNA was prepared from mRNA isolated from phenylhydrazine-treated chicken reticulocytes, and cloned into the tetracycline-resistance gene of plasmid pMB9. Restriction enzyme analysis indicates that there are at least two classes of clones isolated. Nucleotide sequence data have shown that these classes corresponded to the chicken α- and β-globin genes.     

Circularized copies of amplifiable resistance genes from Haemophilus influenzae plasmids.

Tandem repeat amplification of resistance determinants in Haemophilus influenzae plasmids is associated with the occurrence of separate circular DNA molecules. They were demonstrated to represent mono- and multimeric forms of the amplifiable segments of the plasmids which comprise the respective resistance transposons and an additional region designated as an amplification sequence. The latter region mediates the recombinational events involved in amplification. The DNA circles apparently lack the ability to replicate autonomously but most probably provide an effective means for the translocation of resistance genes from one plasmid to another.     

Structure and expression of human globin genes introduced into mouse fibroblasts

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.     

The divergence between human and baboon globin genes.

Complementary DNA (cDNA) was prepared with viral RNA-directed DNA polymerase from purified baboon globin messenger RNA (mRNA). Homologous and heterologous hybrids between human and baboon mRNAs and cDNAs were compared for extent of hybridisation and thermal stability. Higher mRNA inputs to the hybridizations were required to reach saturation in the heterologous cases. The melting temperature of the heterologous hybrid was 5 degrees C lower than the homologous hybrid. Between these two primates, divergence has occurred in the globin gene to a smaller extent than that possible from third position changes in the coding sequences of the divergence of total DNA. Globin cDNA prepared from baboon will not in general be useful as a probe for human globin mRNA or human globin gene sequences.     

Multiple arrangements of the human embryonic zeta globin genes.

Rearrangements which are most readily explained by homologous crossover between misaligned segments of DNA in the region of the human embryonic zeta (zeta) globin genes have been identified in individuals of three different racial origins. These recombination events have resulted in a surprisingly high prevalence of chromosomes with single (0.4%) and triplicated (1.3%) zeta genes with apparently no significant effect on the phenotype.     

Isolation and characterization of cloned human fetal globin genes.

Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clone (NY 1) has a 15.4 kb insert by virtue of an extra 1.4 kb Eco RI fragment at its 5' most end. This clone also has a Kpn I site not present in the other two suggesting it is the product of the gamma gene on the opposite chromosome. Restriction analysis of the three clones indicates that the G gamma and A gamma genes are linked on a single continuous piece of DNA and are separated by 3.5 kb and each contains at least one large intervening sequence of 0.85 kg between the Bam HI and Eco RI sites. These findings in cloned DNA provide direct evidence for linkage and organization of the gamma genes in man.     

Insertion elements on lactococcal proteinase plasmids.

DNA segments of 809 and 808 nucleotides, with 18-base-pair terminal inverted repeats, are present on the proteinase plasmids pWV05 from Lactococcus lactis subsp. cremoris Wg2 and pSK111 from L. lactis subsp. cremoris SK11, respectively. These DNA segments are highly similar: 77% identical nucleotides and both contain an open reading frame that can encode a protein of 226 amino acids. Furthermore, both DNA segments are located downstream of the proteinase maturation gene prtM, but they differ individually in their orientation with respect to the prtM gene. On the basis of the striking similarity between ISS1, an 808-base-pair insertion sequence (IS) from L. lactis subsp. lactis ML3 lactose plasmid pSK08, and the DNA segments of pWV05 and pSK111, we propose that these DNA segments comprise IS elements. The IS elements from strains Wg2 and SK11 were named ISS1W and ISS1N, respectively. On pWV05, ISS1W is flanked on one side by only part of a second IS element, indicating that pWV05 evolved as a deletion derivative of a precursor plasmid that carried at least two IS elements.     

Transfer of human globin genes to erythroleukemic mouse cells.

Thymidine kinase negative (TK-) Friend cells were transformed with recombinant molecules carrying human globin genes and the thymidine kinase gene of herpes simplex virus type 1 DNA. Transformation frequencies of 1 transformant/microgram donor DNA/1 x 10(6) cells were obtained by standard procedures and this was increased 20- to 30-fold by treating recipient cells with dimethyl sulfoxide or glycerol. Transformed cell lines expressed thymidine kinase activity of viral origin as determined by its insensitivity to 0.2 mM dTTP and electrophoretic mobility in polyacrylamide gels. The physical status of donor DNA in the transformed cells was examined in Hirt precipitates and supernatants by Southern blot hybridization and spot hybridization techniques. This analysis showed that most donor sequences were present in a circular or concatenate configuration, but also was suggestive of some donor sequences being integrated into high molecular weight DNA. Expression of human globin genes and particularly the epsilon-globin gene in the transformed Friend cells was studied by Northern blot hybridization analysis.     

Insertion of plasmids into the chromosome of Streptomyces griseofuscus

The results demonstrate a general method for randomly integrating plasmids into the genome by a single crossover between a cloned DNA fragment and homologous DNA in the chromosome. The integrated plasmid is flanked by directly repeated copies of the cloned homologous DNA sequence. Two protocols, "replica plating" and "liquid transfer," yielded strains with integrated plasmids.     

Repetitive DNA sequences near three human beta-type globin genes.

Five repetitive DNA sequences, of average length 259 bp, have been identified in the intergenic regions which flank three human beta-tupe globin genes. A pair of inverted repeat sequences, separated by 919 bp, was found 1.0 kb to the 5' side of the epsiln-globin gene. Each contains a homologous Alu I site. Another repetitive sequence, with the same orientation as the inverted repeat sequence closest to the epsilon-globin gene, lies about 2.2 kb to the 5' side of the delta-globin gene. A pair of inverted repeat sequences, with the same relative orientations as the other pair and separated by about 800 bp, was found about 1.5 kb to the 3' side of the beta-globin gene.     

Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases.

A method of identifying plasmids containing genes responsible for synthesis of nucleotide sugar:lipopolysaccharide glycosyltransferases is described. Hybrid ColE1 plasmids containing random fragments of the chromosome of Escherichia coli K12 were introduced into an indicator strain of Salmonella typhimurium which lacks UDP-glucose:lipopolysaccharide glucosyltransferase I due to an rfaG mutation. Plasmids capable of correcting the transferase defect were identified by their ability to convert the bacteriophage sensitivity pattern of the recipient strain from Ffm-sensitive to Ffm-resistant. Analysis of the lipopolysaccharide of the S. typhimurium/ColE1 hybrid strains and assay of cell extracts defined the new enzyme activities. Two plasmids were identified which carried the rfaG+ gene; one of these plasmids also contained genetic information for a second glucosyltransferase, the E. coli glucosyltransferase II, which normally is not present in S. typhimurium.