Journal of molecular and applied genetics最新文献

We have devised a modification of the Berk and Sharp procedure that allows us to detect the presence of a diverse population of 35S full-length, genomic viral transcripts of cauliflower mosaic virus (CaMV). Using this procedure we have been able to identify and characterize four such 35S transcripts. The first 35S RNA that we have mapped, 35S-1, is a slightly longer than full-length transcript, beginning approximately at nucleotide 7500 and ending at nucleotide 7650 and corresponds to a previously characterized 35S transcript of the CabbB-JI strain of CaMV. The second transcript, 35S-2, begins and ends near nucleotide 8000 on the genome map. The third and fourth 35S RNAs, 35S-3, and 35S-4, have their 5' ends lying, respectively, at approximately nucleotides 1200 and 2100, while their 3' ends lie near nucleotides 1300 and 1900. In addition to the four characterized 35S RNAs, we present evidence for a fifth 35S RNA which may begin and end near nucleotide 5700.

Transfection of monkey cells with recombinant plasmids containing a beta-globin coding region fused to wild-type or deleted simian virus 40 (SV40) early region promoters has allowed an analysis of the transcriptional activity of these promoters in the absence of repression by large T antigen. We find that deletion of the TATA sequence does not reduce the transcriptional effectiveness of the promoter; however, the 5' ends of the transcripts are heterogeneous rather than being restricted to the usual sites. The short GC-rich repeat sequences between nucleotides 37 and 107 and the tandemly repeated 72-bp segment between nucleotides 107 and 250 are each essential for promoter function. The GC-rich repeats are functionally redundant and probably interchangeable, since several subsets of two or three of the GC-rich segments are sufficient. One copy of the 72-bp sequence is sufficient to permit transcription. Moreover, the 72-bp repeat sequences function normally even if they are inverted relative to their normal orientation.

Using specific mutants as a means of identification, the bacterial protein for asparagine synthetase (Asn Syn) was shown to be antigenically and electrophoretically similar to its mammalian counterpart. This observation prompted us to attempt direct transfer of the cloned bacterial gene for the enzyme to mammalian cells. DNA from the replicative form of clone M13 OriC, containing the bacterial gene for Asn Syn, was shown to be capable of causing transformation of Jensen rat Asn Syn- cells to cells capable of growth in Asn-free medium; no prior modification of the bacterial gene was required. This relatively inefficient transformation (20 colonies/micrograms DNA/10(6) cells) was sensitive or insensitive to restriction enzyme digestion of the M13 OriC DNA in complete agreement with the known restriction map of the bacterial gene. Clones of transformed rat cells contained the bacterial DNA, which was amplified if increased levels of the enzyme were demanded and lost if selection was removed. The clones also contained polysomal bacterial RNA and a new protein with properties similar but not identical to those of the bacterial enzyme. The biological significance of this unusual degree of compatibility between the prokaryotic and eukaryotic Asn Syn gene systems is discussed.

The FT37/1 tumor line induced by Agrobacterium tumefaciens on flax epicotyls contains 22-24 copies of the T-DNA encoded nopaline synthase gene per cell. All the gene copies are methylated to some extent but the methylation is not uniform, nor does it reflect the methylation level of the flanking plant DNA. This extensive methylation correlates with an extremely low level of expression of the nopaline synthase gene. Treatment of the tumor line with the in vivo demethylating drug 5-azacytidine at a concentration of 3 X 10(-5) M, resulted in the demethylation of, on average, one copy of the nopaline synthase gene per cell. This demethylation was paralleled by an increase in the transcription of the gene and indicates that cytosine methylation is capable of suppressing the expression of plant genes in vivo.

The structure and expression of the Shrunken (Sh) locus have been examined in several maize strains with mutations at the locus caused by the controlling element Dissociation (Ds). Three of the strains (sh-m6233, sh-m5933, and sh-m6258) are of independent origin, and the fourth (sh-m6795) represents a spontaneous recessive sh allele derived from an Sh revertant of strain sh-m6258. The sh-m6233 and sh-m5933 strains produce undetectable levels of the Sh-encoded sucrose synthetase and very small amounts (less than 1%) of an apparently normal sucrose synthetase mRNA in immature endosperm tissue. Both strains have rearrangements affecting the structure of the locus near the 5' end of the transcription unit. The Sh locus in the related strains sh-m6258 and sh-m6795 is interrupted by an insertion or a rearrangement having one breakpoint in an intervening sequence near the 3' end of the mRNA coding sequence. The 5' end of the gene is transcribed in immature kernels of both mutants, giving aberrant poly (A)+ mRNAs that are homologous to the normal transcript up to the insertion or rearrangement breakpoint and lack homology to the 3' end of the gene. The aberrant transcripts from both strains are translated in vivo and in vitro, yielding 82- and 85-kD proteins that are immunologically related to the 92-kD Sh-encoded sucrose synthetase monomer.

The trifunctional trp-1 gene from Neurospora crassa was cloned by complementation of a phosphoribosylanthranilate isomerase-deficient mutant of E. coli. A 2.7-kb DNA sequence containing trp-1 was determined. Homology of the deduced trp-1 polypeptide sequence to the corresponding E. coli proteins is striking; the order of functional domains within trp-1 is NH2-glutamine amidotransferase-indoleglycerolphosphate synthase-phosphoribosylanthranilate isomerase-COOH (NH2-trpG-trpC-trpF-COOH). Whereas trpF complementing activity can be detected in E. coli, trpC activity is absent. It is likely that translation of trp-1 does not proceed from the proper start site in E. coli; the carboxy terminal portion of the trp-1 polypeptide may be the only portion synthesized. Fusion of a bacterial amino terminus and ribosome binding site to the trp-1 coding region results in expression of trpC as well as trpF activity in E. coli. The locations of several startpoints for trp-1 mRNA synthesis were determined by the S1 nuclease mapping technique. DNA immediately 5' to the trp-1 transcription initiation region does not possess a sequence resembling the canonical TATAAA of eukaryotes.

Gene fusions between the Escherichia coli lacZ gene and DNA segments containing the simian virus 40 early promoter or the mouse mammary tumor virus (MMTV) promoter direct the synthesis of functional beta-galactosidase in Cos 7 monkey cells and mouse Ltk-cells. Enzymatic activity was measured either 72 h after transfection or in stable transformants. The sensitive beta-galactosidase assay was used to measure gene expression and to optimize the efficiency of DNA-mediated transfection. Glucocorticoids stimulated the production of beta-galactosidase when lacZ was fused to the hormonally regulated MMTV promoter.

A contiguous region of cellular DNA sequence, 64 kb in length and representing overlapping cellular inserts from three independent cosmid clones, has been isolated from a representative library of human lung carcinoma DNA partially digested with MboI. Within this region of the cellular genome, v-abl homologous sequences are dispersed over a total region of around 32 kb. These sequences represent the entire v-abl human cellular homologue, are colinear with the viral v-abl transforming gene, and contain a minimum of seven intervening sequences. At least eight regions of highly repetitive DNA sequences have been shown to map in close proximity to c-abl coding sequences. In addition to the major c-abl human locus, three regions of human DNA sequence, corresponding to only portions of the v-abl gene, have been identified. Two of these have been molecularly cloned and shown to be distinct from the primary human c-abl locus. Upon transfection to rat embryo fibroblasts in culture, none of the cosmid DNAs containing v-abl homologous sequences exhibited transforming activity. These findings identify and map a single genetic locus of human DNA, c-abl, representing the complete v-abl homologue, and demonstrate the existence of additional human DNA sequences corresponding to more limited, subgenomic regions of v-abl.

The purpose of this study was to evaluate the usefulness of randomly isolated human cDNA clones as probes for human restriction fragment polymorphisms. Clones were chosen from two human cDNA libraries and tested by hybridization against Southern blots of genomic DNA prepared from nine individuals. Three types of hybridization patterns were seen with the individual clones tested, those representative of single copy genes, others representative of gene families, and one characteristic of a mitochondrial gene. Only two of these cDNA clones revealed any polymorphisms among the individuals tested: pDK-08 and RW12, both representative of gene families. Both revealed independent polymorphisms with several of the restriction enzymes tested, and inheritance of these polymorphisms as Mendelian alleles was verified by human pedigree analysis. The results are discussed with regard to the usefulness of such cDNA clones in revealing polymorphisms in human genetic analysis.