Journal of molecular and applied genetics最新文献

A general method for creating somatic cell hybrids which maintain chromosome segments by selection is described. To extend the technique of metaphase chromosome transfer to regions of the genome which do not carry a selectable marker, a retroviral vector has been employed to introduce the dominant selectable neo gene at many genomic locations in a population of murine cells. We have used such cells as donors in metaphase chromosome transfer experiments in which hamster and monkey cells were used as recipients. Cells acquiring a transferred chromosomal segment containing the neo gene were selected by growth in the presence of the drug G418. Hybridization to cloned interspersed repeat DNA sequences of the mouse was used to estimate the proportion of mouse DNA in each transferent. These experiments indicate that transferents produced in this manner contain 0.01 to 1.0% of the mouse genome. To analyze the organization of the DNA transferred to each recipient, we used Southern transfer hybridization of DNA from each transferent to a cloned mouse interspersed repeated DNA probe which did not cross-hybridize to hamster or monkey DNA. We found that each primary transferent gave a unique pattern of restriction fragments hybridizing to this probe. Secondary transferents from two independent primary transferents were compared by this technique. Each set of secondary transferents exhibited a hybridization pattern which resembled closely that of the primary transferent from which it was derived. However, a number of hybridizing DNA segments present in each primary transferent were absent in some of the secondary transferents. These results are most compatible with the view that an intact segment of the mouse chromosome surrounding the integration site of the retroviral vector can be transferred by the techniques used in this study. We believe this technique will be generally applicable to cells from many species, and will allow the study of chromosome regions previously refractory to analysis by chromosome transfer techniques.

Antibodies reactive against mammalian RNA polymerase II were isolated by affinity chromatography. Serum from a goat immunized with Drosophila RNA polymerase II was passed over a column containing covalently coupled calf thymus RNA polymerase II, and reactive antibodies were eluted at low pH. The affinity-purified antibody was reactive against two large subunits (Mr = 140,000-220,000) and four small subunits (Mr = 16,000-35,000) of RNA polymerase II purified from calf, human, chicken, and Drosophila, as well as two large subunits of wheat germ RNA polymerase II. These findings confirm that RNA polymerase II subunits share conserved antigenic determinants over wide evolutionary distances. The affinity-purified antibody was also used to investigate the presence and structure of RNA polymerase II in human cell extracts capable of carrying out promoter-selective transcription in vitro. The subunit structure of RNA polymerase in the extract appears to be the same as that of purified human RNA polymerase II. In both cases, the human enzyme differs from the calf thymus enzyme in that it contains a large (Mr = 220,000) form of the largest subunit.

Two potential shuttle vectors which contained the identical herpes simplex virus type 1 (HSV-1) defective particle DNA (dDNA), but prokaryotic DNA of different origins, were examined for their stability when propagated in eukaryotic cells, and for their efficiency as shuttle vectors. Each chimeric molecule contained a 9.5 kilobase-pair (kb) EcoRI fragment (HSV12-7) representing a single unit of a class I HSV-1 dDNA. This dDNA was cloned into the bacteriophage lambda (lambda) vector lambda gtWES X lambda B' to create a 45.3 kb chimeric molecule (lambda gtWES::12-7), and into the plasmid vector pBR325, resulting in a 15.5 kb recombinant DNA molecule (pBR325::12-7). Each of these DNA molecules was transfected independently into African green monkey kidney cells which were then infected with wild-type HSV-1 helper virus. Both chimeric molecules were replicated and packaged into HSV-1 virions. However, regions of the lambda gtWES::12-7 chimeric DNA were rapidly deleted and rearranged, whereas the plasmid/HSV-1 DNA molecules were less rearranged. No intact lambda gtWES::12-7 DNA was recovered from HSV-1 virions as detected by infectivity of in vitro packaged DNA. However, pBR325::12-7 DNA isolated from HSV-1 virions was able to transform E. coli to ampicillin resistance. These results suggest additional considerations when designing single units of HSV-1 dDNA for use as vectors to accommodate large fragments of DNA.

We have selected alfalfa suspension cell lines that are 20- to 100-fold more resistant than wild-type cells to the nonselective herbicide L-phosphinothricin, a mixed competitive inhibitor of glutamine synthetase (GS). GS enzyme levels are three- to sevenfold elevated in the variant cell line. Partial amino acid sequences of two cyanogen bromide cleavage peptides have been determined on GS purified from the variant cell line and used to confirm the identity of a 276-base pair GS cDNA clone. Southern blot analysis of wild-type and variant cell DNAs using this cDNA as a probe indicate that resistance to L-phosphinothricin is a consequence of a four- to 11-fold amplification of one GS gene resulting in about an eightfold increase in mRNA levels, and an increased enzyme synthesis sufficient to overcome the toxic effects of the inhibitor.

In several species of Rhizobium the three genes which encode the nitrogenase complex are separated into two operons, nifH and nifDK. We have mapped the transcriptional promoter sites for these two operons from R. japonicum USDA strain 110 by S1 protection analyses using bacterial RNA isolated from soybean nodules. Transcription of the nifDK operon is initiated at a site located 46 nucleotides upstream of the proposed translation initiation codon. nifH transcription initiates predominantly at a site 152 nucleotides upstream of the proposed translation initiation codon. An additional minor start for nifH is found 35 nucleotides downstream of the major initiation site. The nucleotide sequences of these promoter sites are presented. Comparison of the major R. japonicum nif promoter sequences reveals a high degree of sequence homology with the conserved sequences clustered in the -11 to -15, -21 to -25, -27 to -31, and -38 to -41 regions. Conservation is also observed for the -11 to -15 and -21 to -25 regions with the R. meliloti nifHDK, a cowpea Rhizobium nifH, and even Klebsiella pneumoniae nif promoters. The -27 to -31 and -38 to -42 region homologies are not conserved with the other known nif promoters. These results are discussed in light of models for nif promoter activation.

A naturally occurring variety of soybean Glycine max cv. Keburi, which lacks the alpha'-subunits of the 7S seed storage protein (beta-conglycinin), was recently described by Kitamura and Kaizuma. Keburi beta-conglycinins contain a normal complement of alpha-, beta-, and gamma-subunits that accumulate in a temporally and spatially regulated manner comparable to that of the standard cultivar Provar. Poly(A)+ RNA isolated from mid-to-late maturation stage Keburi seeds was translated in vitro, yielding products equivalent to those of Provar poly(A)+ RNA except that Keburi mRNA did not produce the pre-alpha'-subunit polypeptide. The basis of the Keburi phenotype was determined by examining genomic DNA using Southern blot hybridization. Restriction fragments isolated from a cloned 11.5 kb EcoRI fragment of genomic DNA containing a normal alpha'-subunit gene (Gmg 17.1) were used as hybridization probes. Sequences far upstream of the alpha'-subunit gene were present in both Provar and Keburi cultivars. However, hybridization reactions with probes from within the gene demonstrated that a deletion had occurred in Keburi DNA beginning immediately 5' to the alpha'-subunit gene represented on this 11.5 kb fragment. The deletion extends through most of the coding sequences, producing the Keburi phenotype.

The nucleotide sequence of a tumor morphology gene, tmr, from the Agrobacterium tumefaciens Ti-plasmid, pTiA6NC, and its flanking 5' region was determined by M13 "dideoxy" procedures. The DNA sequence reveals an open reading frame capable of encoding a 240 amino acid protein. We have identified the polyadenylated transcript initiation and termination sits by S1 nuclease mapping. The extent of the sequence required for transcription 5' to the start of transcription has been delimited by two transposon insertions. The first of these maps at -- 121 with respect to transcription initiation and results in the wild-type phenotype, the second insertion maps at about -85 and results in a tmr phenotype.

The expression of a protein-coding DNA fragment in a microorganism such as Escherichia coli requires that the exogenous DNA segment be inserted precisely in phase with bacterial translation initiation signals. We report the construction of derivatives of the M13 vectors M13mp7 and M13mp701 in which a HindIII site has been inserted, within the N-terminal section of the beta-galactosidase gene, in all three phases of translation. These vectors may thus be used for the expression, under the control of the lac promoter and translation initiation signals, of protein coding DNA segments flanked by a HindIII site. In all cases the insertion of a DNA fragment can be recognized by the abolition of beta-galactosidase activity. These vectors have been used to direct the expression, in E. coli, of a cDNA segment coding for the rabies virus surface glycoprotein. The proteins produced have been shown to react with antisera raised against authentic rabies glycoprotein.

A genomic library of Phaseolus vulgaris DNA was screened using a lectin cDNA clone that includes the entire coding region to obtain a lambda clone containing the corresponding nuclear lectin gene. Comparison of the cDNA and genomic nucleotide sequences showed that the gene has no intervening sequences. The 5' untranslated region was found to be only 10-16 base pairs long by S1 nuclease mapping. The lectin transcriptional unit is flanked by sequences which are exceptionally high (72-75%) in A + T content and its 5' flanking region contains potential transcription control elements.

The promoter region of the CaMV inclusion body protein gene was modified for use in chimeric gene fusions. The modified promoter was used to construct a selectable marker for plant transformation based on the Tn 5 kanamycin resistance gene. This chimeric selectable marker was introduced into plant cells using oncogenic and deoncogenized strains of Agrobacterium tumefaciens. Both types of transformation produced kanamycin-resistant cell lines. The resistant cell lines derived from the deoncogenized strains were used to regenerate shoots. A second type of selection based on the ability of octopine synthase to detoxify aminoethyl cysteine was also used to select transformants in both oncogenic and nononcogenic transformation.