Journal of molecular and applied genetics最新文献

We have examined the nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase from soybean. One member of this gene family, designated SRS1, has been isolated from a soybean DNA library constructed in the lambda vector Charon 4A. The complete nucleotide sequence and structure of this gene including its two introns and portions of the 5' and 3' flanking sequences were determined. The first exon encodes the entire transit peptide (55 amino acids) and the first 2 amino acids of the mature sequence. Based on analysis of the nucleotide sequence, we concluded that the precursor of the soybean small subunit consists of 178 amino acids. A gene-specific probe for SRS1 was used to show that this gene is transcribed and that steady-state levels of its transcript are strongly light regulated. S1 nuclease mapping was used to locate the potential start of transcription in the sequence and showed that the small subunit gene contains a cap site, TATA box, and -80 sequence, which match the consensus animal sequences. The mature SRS1 small subunit polypeptide of 123 amino acids contains 30 and 34 amino acid replacements relative to the previously determined amino acid sequences from pea and spinach, respectively. Southern blotting of restriction digests of soybean nuclear DNA and data on the complete structure of SRS1 suggest that a multigene family of at least 10 members encodes the RuBP carboxylase small subunit in soybean. Quantitative evolutionary comparison of the soybean small subunit sequence for SRS1 and the pea small subunit sequence suggests that these two genes diverged long before the divergence of pea and soybean.

A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are killed by the antibiotic G418, those that acquire and express neo continue to grow in the presence of G418. In the course of the selection, neo DNA becomes associated with high molecular weight cellular DNA and is retained even when cells are grown in the absence of G418 for extended periods. Since neo provides a marker for dominant selections, cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.

Cadmium and other heavy metals induce metallothionein synthesis in mammalian cells. We have cloned a mouse metallothionein-I gene from a cadmium-resistant line of Ltk- cells and inserted it into viral and plasmid SV40 vectors. These recombinant molecules were introduced into cultured monkey cells and the expression of the foreign metallothionein gene was analyzed, at the RNA level, in the presence and absence of cadmium. The results show that the cloned gene retains its cadmium inducibility and that the regulation of this gene occurs primarily at the transcriptional level.

The gene coding for the glycolytic enzyme triose phosphate isomerase (TPI1) was isolated from a yeast library in the shuttle vector pYE13. Selecting for a deletion mutant of the plasmid which enhances expression of the otherwise dormant yeast gene in E. coli facilitated the identification of the coding region. The DNA sequences of the wild type and mutant genes were determined by chemical methods. The 5' flanking region of the wild-type TPI1 resembles the analogous regions of the yeast genes coding for two other glycolytic enzymes. The sequence of the deletion mutant indicates that, upstream from -65 in the 5' flanking region, 3.3 kilobases have been lost from entirely within the yeast insert. The mutation reduces enzyme activity by tenfold in yeast, and its implications for the expression of the gene in yeast and E. coli are discussed. The amino acid sequence deduced from the nucleotide order is consistent with the electron density map of the protein as well as the sequence of its N-terminal 16 amino acids and amino acid composition. The amino acid sequence is approximately 50% homologous with the triose phosphate isomerases from rabbit, chicken, and coelacanth and 37% homologous with the Bacillus stearothermophilus enzyme. Residues which are thought to be catalytically important are conserved.

Pairs of defective cauliflower mosaic virus genomes coinoculated in plants are rescued at high frequency by homologous, intergenomic recombination events. Defective genomes may be rescued by one of at least two general types of recombination mechanisms. One mechanism appears to operate on linear DNA molecules with complementary sticky ends. Recombination of this type can be explained by the formation of mixed dimers (or concatamers) which can be resolved into normal genomes by a single crossover event. Another type of recombination takes place among viral DNA molecules which do not have complementary sticky ends. Rescue of this type yields normal viral DNA molecules which can be resolved only by the equivalent of a double crossover or internal gene conversion event between pairs of infecting molecules. Co-infection of plants with pairs of defective genomes derived from different viral isolates results in the formation of hybrid viral genomes. Analysis of these hybrids reveals that recombination can take place at different sites in the viral genome.

A transposon (Tn5)-induced mutant (strain ANU437) of Rhizobium trifolii was isolated in which no water-soluble exopolysaccharide (EPS) could be detected. This mutant was also incapable of forming nitrogen-fixing root nodules on clover plants. Molecular cloning has demonstrated that the Tn5 transposon was responsible for both of these mutant phenotypes and that there is a direct correlation between EPS synthesis in this bacterial strain and its ability to carry out symbiotic nitrogen fixation. In the mutant ANU437, Tn5 was located in a 9.4-kb EcoRI fragment that was cloned into the amplifiable plasmid pBR322. The recombinant plasmid was used as a hybridization probe to isolate the corresponding wild-type DNA sequence of R. trifolii from a lambda Charon 28 genomic clone bank. This DNA sequence was subcloned into the broad host range conjugative plasmid RP4 and introduced into the Escherichia coli strain RR1. It was then transferred to the mutant ANU437 by conjugation. The acquisition of the wild-type DNA sequence by the mutant ANU437 resulted in the restoration of its ability to synthesize normal levels of EPS and to form nitrogen-fixing nodules on white and subterranean clovers.

After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes. Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes. ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3). A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe. ISRm1 is present at least ten times in the R. meliloti genome but is not present in any other R. meliloti strains, E. coli strains, or Rhizobium species tested. We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R. meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment. The resulting strain was Fix-.

Cloned bovine papilloma virus (BPV) DNA induces cellular transformation when introduced into mouse cells growing in culture; the transferred viral DNA replicates as an extrachromosomal, closed circular element. BPV DNA is therefore an inviting replicon to construct a "shuttle" vector that can replicate in both mammalian cells and E. coli. Although BPV DNA devoid of bacterial plasmid sequences has been successfully employed to reintroduce cloned genes into rodent cells, construction of a true shuttle plasmid has been hampered by a disruptive influence of bacterial plasmid sequences that appear to block cellular transformation when included on the transferred molecule. We constructed a molecule, pGP, containing the transforming region of the BPV genome, the rat growth hormone gene, and bacterial plasmid pBR 327, and have found unexpectedly that the intact molecule can induce cellular transformation of mouse cells at high efficiency despite the presence of bacterial sequences in the transferred plasmid. A similar plasmid without the growth hormone segment does not transform mouse cells. The pGP molecule replicates as a stable plasmid in both mouse cells, where there are 30 to 80 monomer episomes per cell, and E. coli, and may be shuttled back and forth unaltered between the two kinds of cells. The growth hormone gene is transcribed in the mouse cells and gives rise to a transcript that is longer than authentic rat growth hormone mRNA and does not appear to be regulated by glucocorticoids. When pGP is cotransferred into mouse L-cells with herpes simplex virus tk gene, it appears to integrate, and free monomer episomes are not observed.

We have constructed a eukaryotic expression vector designed to express a gene under late SV40 transcriptional control. From this chimeric plasmic-SV40 vector, virtually all the sequences which code for the major capsid protein VP1 have been deleted and instead, the human fibroblast interferon beta 1 cDNA gene has been inserted. After transfection of monkey cells with this recombinant, substantial quantities of human beta 1 interferon (up to 2 x 10(-4) IU/ml) were excreted in the culture medium. Transfection of nonpermissive mouse L cells or rat cells yielded virtually undetectable quantities of human beta 1 interferon (5 x 10(3) to 10(4) times less than that in monkey cells). The recombinant SV40 vector may serve as a model vehicle for the efficient expression of other eukaryotic genes and might also be used as a direct screening vector for cloning of eukaryotic or prokaryotic cDNA genes.

We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.