Journal of molecular and applied genetics最新文献

DNA isolated from hairy roots incited on Daucus carota by Agrobacterium strains harboring the Ri plasmid was probed with Ri plasmid fragments to identify plasmid sequences transferred and integrated into the plant genome. One hairy root line was incited by an A. rhizogenes natural isolate strain harboring two large plasmids in addition to the Ri plasmid. DNA from hairy roots incited by this strain contained a total of five nonidentical copies of T-DNA which together comprised a T-DNA complement representing a 34-42 kb segment of the Ri plasmid. A second hairy root line was incited by a Ti plasmidless A. tumefaciens strain into which the Ri plasmid had been conjugated. Hairy root DNA incited by this strain contained four identical T-DNA copies 17-18 kb in length and one truncated copy. T-DNA borders occurred at preferential sites of the Ri plasmid. T-DNA structure in these hairy root lines is similar to T-DNA structure in tumors incited by Ti plasmids of A. tumefaciens.

Globin structural genes from a murine erythroleukemia cell line were analyzed by Southern blot hybridization of genomic DNA and after isolation of cloned globin genes from a genomic library. The globin genes isolated from the erythroid cell line did not differ, when analyzed by extensive restriction endonuclease digestion, from globin genes isolated from nonerythroid cells. No gross structural differences were seen between murine erythroleukemia globin genes, either before or after hexamethylene bisacetamide (HMBA)-mediated erythroid differentiation, and globin genes from normal mouse liver DNA. Whereas the murine erythroleukemia genome hybridizes extensively to cloned Friend leukemia virus probes, there was no evidence of viral integration into sequences adjacent to the globin genes.

Chimeric genes have been constructed by inserting foreign gene sequences in the early region of SV40. The genes contained the first exon of the SV40 large T gene with 180 bp of its intron and either the third exon of the rat preproinsulin gene II with 488 bp of its large intron or the third exon of the mouse beta globin gene with 63 bp of its intron. The chimeric genes contained a 5' splicing site (SS) from SV40 and a 3' SS from the inserted gene. Both the preproinsulin and the globin insertions contained a polyadenylation signal. The SV40 early poly(A) addition signal was also retained. High-titer virus stocks were obtained when the recombinants, which contained SV40 origin of replication and the entire late region, were used to transfect a cloned line of COS cells (COS-M6). These stocks typically contained no detectable wild-type virus. RNA mapping demonstrated the following: (a) The SV40-rat preproinsulin chimeric RNA was initiated at the SV40 early promoter, spliced from the SV40 5' SS to the rat preproinsulin 3' SS, and polyadenylated solely at the SV40 poly(A) addition signal. (b) The SV40-mouse beta globin chimeric RNA was initiated at the SV40 early promoter, spliced from the 5' SS to the mouse beta globin 3' SS, and polyadenylated at the mouse beta globin poly(A) site. The chimeric RNAs were overproduced, owing to low levels of T antigen in the COS-M6 cells, which did not completely repress transcription from the early region. Fusion proteins of 15,500 molecular weight resulted from expression in vivo of the SV40-rat preproinsulin chimeric gene and of 11,500 molecular weight for the SV40-mouse beta globin chimeric gene. The molecular weights of the proteins suggested that they were initiated at the early SV40 AUG and that translation continued across the chimeric splice sites. The chimeric proteins were also overproduced.

We have molecularly cloned and sequenced cDNA to the transcript of H-ras-1, the transforming gene of the T24 human bladder carcinoma cell line. The transcript derives from at least five exons in the H-ras-1 gene, and RNA splicing occurs at sites typical of exon-intron junctions. T24 H-ras-1 RNA has an AUG-initiated open reading frame of 567 nucleotides, which can encode a protein of mass comparable to the apparent molecular weight of the T24 H-ras-1 gene product. The T24 H-ras-1 gene product is nearly identical to v-H-ras p21, the transforming protein encoded by the genome of Harvey sarcoma virus. We discuss the implications of this sequence conservation in the structure-function relationships of ras proteins.

Five distinct cDNA clones for the major chlorophyll a/b binding protein (Cab) of the petunia thylakoid membrane light-harvesting complex have been characterized. The nucleotide sequences of the polypeptide coding regions are similar between clones; however, the 3'-untranslated regions are divergent. Analysis of petunia nuclear DNA through genomic hybridizations and characterization of cloned nuclear fragments indicates there are at least sixteen genes for the major Cab protein. These genes can be classified into at least five small multigene families on the basis of their homology to the different cDNA clones. Two families contain only two genes each and in both cases the genes are closely linked in the genome. In one case, one gene is in an inverted orientation with respect to the other such that the 5' ends are adjacent; in the other case, the two genes are in a tandem array. Both genes from one of these families are transcribed in green leaves. The genes belonging to separate nuclear gene families encode different polypeptide components of the major Cab protein.

The E. coli lambda lysogen, OR1263, carries the fusion pR-cro-tR1-IS2-gal. The gal promoter is deleted and gal expression from pR, in the absence of the lambda antitermination factor N, is blocked by the efficient transcription terminator in IS2. Selection for Gal+ yields strains deleted for the IS2 terminator and various portions of the lambda chromosome. Analysis of these deletions reveals the following: (a) The lambda tR1 terminator is about 50% efficient. (b) In two deletions sequenced, DNA loss occurred as a result of homologous recombination between a 2- or a 4-base pair repeat. (c) By measuring the ability of lambda N product to suppress the polarity of a gal ochre mutation, we demonstrate that the N utilization site in the lambda pR operon lies between tR1 and cro. (d) The level of Cro repressor synthesized by a single copy prophage is sufficient to repress the cI maintenance promoter, prm, but is inadequate to inhibit pR.

Restriction endonuclease-digested deoxyribonucleic acid (DNA) from 17 slow-growing Rhizobium strains was hybridized with 32P-labeled DNA of the Klebsiella pneumoniae nitrogenase structural gene (nifKDH) region. Sixteen of these strains contained two or more fragments that were homologous to K. pneumoniae nifKDH after cleavage with EcoRI or HindIII. Hybridization with nifKDH subclones revealed that most of the Rhizobium fragments were homologous to the HindIII-EcoRI portion of nifKDH (corresponding to the nifDH region), although fragments homologous to the EcoRI-HindIII portion of nifKDH (corresponding to the nifK region) were also present. Comigrating nif-homologous restriction endonuclease fragments were observed for strains isolated from different geographic locations and from nodules of different plant species. Nearly identical hybridization patterns were obtained for five R. japonicum strains which clearly differed in the pattern of restriction endonuclease fragments from their chromosomal DNA. This indicates that there is a high degree of conservation of DNA sequence surrounding the region of nif homology in these strains.

Although the human rDNA gene family is organized in clusters of tandem repeats on five pairs of acrocentric chromosomes, all rDNA genes have undergone a "concerted" evolution resulting in a homogeneous population of genes. Two steps are necessary for a variant to spread to all rDNA genes: dissemination of the variant to all genes in a cluster; and exchange of rDNA genes between nonhomologous chromosomes. To study the organization of rDNA genes, a restriction fragment length polymorphism in the spacer region adjacent to the 28S gene was examined in somatic cell hybrids in which individual human acrocentric chromosomes could be isolated. Human DNA cut with BamHI and analyzed by Southern hybridization yields two to four major bands that hybridize to a 32P-labeled cloned fragment of the 28S gene. Hybrids containing single human acrocentric chromosomes do not recapitulate the parental patterns, but frequently have only one of the parental bands. The data suggest that the quantitative distribution of spacer length variants differs among the human acrocentric chromosomes in hybrids. The frequently observed homogeneity of the rDNA variants on individual acrocentric chromosomes in hybrid cells may reflect the individual rDNA clusters in the parental cell or may be a result of unequal crossing over in the hybrid cell.

The synthesis of fiber protein in CV1 (monkey) cells abortively infected with human adenovirus serotype 2 (Ad2) is at least 100-fold less than the synthesis of fiber protein in CV1 cells productively infected with a host range mutant of Ad2 (Ad2hr400) or coinfected with Ad2 plus simian virus 40. However, the amount of fiber mRNA present in the cytoplasm of abortively infected CV1 cells is only 5- to 10-fold less than that in productively infected CV1 cells. Whereas fiber mRNA in abortively infected CV1 cells is utilized poorly as a template for synthesis of fiber protein in vivo, fiber mRNA from abortively infected CV1 cells serves just as efficiently as a template for fiber synthesis in vitro as fiber mRNA from productively infected cells. This was observed both in a nuclease-treated rabbit reticulocyte lysate to which purified fiber mRNA or cytoplasmic ribonucleoprotein complex was added as exogenous template for fiber synthesis, and in S10 extracts of infected CV1 cells utilizing endogenous message as a template. Since translation initiation inhibitors did not diminish synthesis of fiber in S10 extracts of abortively infected CV1 cells, fiber mRNA probably is associated with ribosomes in abortively infected CV1 cells. This conclusion was supported by Northern blot analysis, which showed that in both abortively and productively infected CV1 cells, the same proportion of cytoplasmic fiber mRNA cosedimented with polyribosomes. Although the possibility of extremely rapid fiber turnover in abortively infected monkey cells cannot be rigorously excluded, preliminary data suggest that this is not the case. Thus, these results may imply that translation of the fiber message in abortively infected monkey cells is blocked after formation of the mRNA-ribosome complex.

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.