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.

Napins are a family of small, basic storage proteins synthesized in Brassica napus (rapeseed) embryos during seed maturation. Cultured embryos also synthesize napins but require exogenous abscisic acid (ABA) to maintain high accumulation rates. We synthesized cDNA from total RNA of embryos cultured on a medium containing ABA, and cloned it into the Pst1 site of pBR322. Two clones containing napin cDNA sequences selected by differential colony hybridization using [32P]cDNA probes from embryos grown with or without ABA were analyzed. These clones, pN1 (insert size = 583 bp) and pN2 (insert size = 739 bp), contained cDNA from two different napin mRNAs. The mRNAs to which they hybridized were found to encode a 21,000-dalton polypeptide that was immunoprecipitated by antibodies to mature napin (subunits of 9,000 and 4,000 daltons). The cDNA clones hybridized to an 850-base mRNA. Nucleotide sequencing demonstrated 95% homology between pN1 and pN2 cDNA inserts and predicted a precursor polypeptide of 178 amino acids, consistent with the 21,000 dalton in vitro translation product. Comparison of the deduced amino acid sequence with published amino acid compositions of mature napin subunits suggests that both the large and the small subunits are present in one precursor polypeptide, and that other regions of the precursor are removed during processing.