Journal of molecular and applied genetics最新文献

The v-src gene was removed from Rous sarcoma virus DNA and replaced with either a cDNA or genomic clone of human alpha-chorionic gonadotropin. Transfection of the recombinant retrovirus genomes into normal chicken fibroblasts produced nontransforming recombinant virus in high titer. Neither helper virus nor selective conditions were needed. Cells infected with the recombinant viruses expressed RNA containing the gonadotropin sequences in levels equivalent to those of term human placenta (approximately 0.5% of poly A+). Essentially every fibroblast in the culture was infected; the infected cells contained approximately one recombinant provirus each. The gonadotropin intervening sequences were removed precisely from the recombinant genomes that contained them, creating recombinants carrying a perfect cDNA copy of the original genomic insert. However, the intervening sequences were removed inefficiently such that several viral replicative cycles were necessary before all genomes had been processed completely. The implications of these observations to the transduction of viral oncogenes and the creation of processed pseudogenes are discussed.

We have studied the initiation of human ribosomal RNA synthesis in vitro using a cell-free polymerase I transcription system derived from HeLa cells and cloned human ribosomal DNA containing the site of initiation for ribosomal RNA synthesis. Mapping of the RNA products by run-off assays and high-resolution S1 nuclease analysis indicated that transcription in vitro initiates at a unique site and that the RNA has the same 5' terminus as in vivo precursor ribosomal RNA isolated from nuclei of HeLa cells. To provide additional evidence for the initiation site of ribosomal RNA transcription, dinucleoside monophosphates complementary to a sequence on the template have been used as primers for ribosomal RNA synthesis. When the concentrations of the four nucleoside triphosphates in the transcription reaction were reduced to 10 microM, [alpha-32 P]GTP was no longer incorporated into the run-off transcript. Under these conditions, we then tested a variety of dinucleotides for their ability to initiate promoter-specific RNA synthesis, and found that GpC exhibited maximum stimulation. We have also determined that the human cell-free system exhibits a significant degree of template specificity and is able to transcribe ribosomal DNA derived from human and rhesus monkey cells but not from mouse cells.

We developed a method for conveniently positioning foreign DNA at many preselected sites in the adenoviral genome by a combination of in vitro and in vivo recombination. Using this technique, we constructed a set of recombinant viruses that contain the SV40 A gene downstream from the adenovirus tripartite leader. One of these hybrid viruses, Ad-SVR26, contains the A gene close to and downstream from both the major late promoter and the first segment of the tripartite leader. The transcripts encoded by the inserted SV40 DNA are highly overproduced in infected cells; they initiate at the adenoviral late promoter and terminate at the SV40 polyadenylation site. Several novel splice acceptor sites in the SV40 sequences are used in the processing of the primary transcript to produce six different species of spliced RNA. The synthesis of T antigen in Ad-SVR26-infected cells requires the use of novel AUG initiation codons present within the SV40 coding region or adenoviral sequences that normally form part of the intron between the first and second segments of the tripartite leader. The level of T antigen expression is not as high as the level of mRNA production. The usage of these new AUG triplets or the absence of the complete adenovirus tripartite leader sequence may account for the low efficiency of translation.

DNA sequences responsible for the development and maintenance of symbiotic nitrogen fixation have been identified and isolated from Rhizobium trifolii. Symbiotically-defective strains were generated by random mutagenesis with the transposon Tn5. The defective genes which give rise to the mutant phenotype have been cloned into bacterial plasmids and used as hybridization probes to isolate the corresponding wild-type genes from a clone bank of R. trifolii DNA. Symbiotic genes cloned in this manner are able to correct the lesion caused by the insertion of the transposon in their respective mutants and so restore the nitrogen fixation phenotype. The correction of the mutation is shown to occur by two distinguishable mechanisms--either by complementation or by homologous recombination. This approach provides a reliable method for isolation and mapping of bacterial DNA sequences involved in symbiotic nitrogen fixation.

The SV40 early region promoter, previously localized to the DNA segment bounded by the HpaII and HindIII restriction sites (nucleotides 346 and 5171), was further defined by construction of an extensive set of deletions within this region and measurement of their effects on (a) viral DNA replication, (b) virus multiplication and the ability to complement early and late mutations, (c) transformation of rat cells, (d) large T antigen formation, and (e) the location of the 5' ends of early mRNAs. One set of mutations is represented by deletions that begin at the HpaII site and extend unidirectionally for varying lengths toward the BglI site at ori. A second set of mutants contains deletions that start at ori and extend unidirectionally for varying lengths towards the HpaII site. A third set of mutants, with deletions or duplications of various lengths and boundaries, lie between the HpaII and BglI sites. Our studies indicate the following. (a) Ori, the sequence needed for initiating SV40 DNA replication, extends from the sequences needed for initiating SV40 DNA replication, extends from the sequences needed to bind T antigen to the palindrome in site II to nucleotide 34, the late region edge of the AT block. Flanking sequences adjacent to the AT block facilitate DNA replication. (b) The SV40 early region promoter comprises two functionally distinct nucleotide sequence elements. One is flanked by nucleotides 5231 and 107, and contains the RNA initiation sites at nucleotides 5231-5237, a positioning element resembling the TATAAATA consensus sequence about 20-25 nucleotides upstream, and an RNA polymerase II recognition sequence contributed by short GC-rich sequences clustered between nucleotides 35 and 107; we refer to this as the RNA polymerase II interaction site. The second distinct sequence element is contained within each of two 72-bp segments located between nucleotides 107 and 250; the behavior of this element suggests that it may influence the accessibility of RNA polymerase II for the interaction site or the efficiency of RNA chain initiation. Large T antigen binding sites I, II, and III overlap with the putative RNA polymerase II interaction site; since large T antigen does not prevent elongation of RNA transcripts initiated upstream, T antigen probably represses early region expression by preventing RNA polymerase II binding to the promoter.

A cDNA segment coding for rabbit beta-globin has been inserted at different locations in the early region of simian virus 40 (SV40). The inserted sequences in these recombinants are transcribed from the SV40 early region promoter, and the primary transcripts are processed to mature mRNAs using viral intervening sequences and the early region polyadenylation site. After infection with various recombinants, beta-globin polypeptide is synthesized only when the beta-globin translation initiation codon is the first AUG in the messenger RNA sequence. When the early region transcripts contain the beta-globin cDNA sequence 3-proximal to the small t antigen coding sequence, beta-globin synthesis is not detectable. However, these recombinants produce small t antigen and abbreviated forms of large T antigen.

The chloroplast enzyme, ribulosebisphosphate carboxylase, consists of large subunit polypeptides encoded in the chloroplast genome and small subunit polypeptides encoded in the nuclear genome. Cloned DNA complementary to the small subunit mRNA hybridizes to a single RNA species of 900-1000 nucleotides in both total and poly(A)-containing RNA from leaves of Pisum sativum, but does not hybridize to chloroplast RNA. Small subunit cDNA hybridizes to at least three RNA species from nuclei, two of which are of higher molecular weight than the mature mRNA. A cloned large subunit DNA sequence hybridizes to a single species of Pisum chloroplast RNA containing approximately 1700 nucleotides, but does not hybridize to nuclear RNA. The light-stimulation of carboxylase accumulation reflects increases in the amounts of transcripts for both subunits in total leaf RNA. Transcripts of the small subunit gene are more abundant in nuclear RNA from light-grown leaves than in that from dark-grown leaves. These results suggest that the stimulation of carboxylase accumulation by light is mediated at the level of either transcription or RNA turnover in both nucleus and chloroplast.

Three strains of Rhizobium japonicum were examined for the presence of interspecific conserved plasmid-borne DNA sequences and the location of their nif DNA sequences. Strains 61A76 and 110, which both form effective (nitrogen fixing) nodules on soybeans show very low (24%) total DNA sequence homology with each other; strain 61A76 contains one plasmid, and strain 110 contains no identifiable plasmids. Strain 61A24 which forms ineffective nodules on soybeans shows relatively high (50%) sequence homology to strain 110 and contains two plasmids. While the total sequence homology between purified plasmid DNA isolated from strains 61A76 and 61A24 appears limited, heterologous hybridizations between plasmids and total DNAs from these strains and DNA from strain 110 suggest that several sequences which are plasmid-borne in one strain can occur in the chromosome or in very large plasmid(s) in the other strains. The plasmid isolated from strain 61A24 exhibits some homology with several SmaI fragments of an octopine Ti plasmid from Agrobacterium tumifaciens, whereas the 61A76 plasmid shows no homology to this Ti plasmid. DNA sequences from five strains of Rhizobium japonicum were found to hybridize with pSA30, a plasmid carrying the Klebsiella pneumoniae nif-KDH genes, but these sequences were not located on the isolated plasmids. The organization of these genes appears to be similar in strains 110 and 61A24, but is different in strain 61A76.

We have constructed a series of tk+ cell lines by DNA-mediated gene transfer to correlate chromosomal behavior and DNA sequence alterations associated with reversion to the tk- phenotype. Tk- revertants were selected from each of four well-characterized transformed cell lines containing the viral tk gene and multiple human growth hormone genes (HGH). Tk- colonies were analyzed for the presence of tk and HGH sequences by blot hybridization to restriction endonuclease cleaved DNA. Revertants were further characterized by detailed karyotype analysis and hybridization in situ. Blot hybridization of forty tk- revertants indicates that over half of the revertants delete all of the transforming DNA from the recipient chromosome. In fifteen additional revertants, significant deletion has occurred, although transforming DNA is retained. The analysis of chromosomes by Giemsa banding together with hybridization in situ reveals that the deletion of transforming DNA is never associated with loss of an entire chromosome. Reversion to the tk- phenotype, therefore, seems to involve discrete deletions of transforming DNA without apparent chromosome loss. In this restricted set of mutants, it thus seems crucial to maintain the diploid chromosomal complement.