Journal of molecular and applied genetics最新文献

A strain of Bacillus subtilis has been developed and tested for use as the host component of the B. subtilis HV2 system. The strain was constructed incorporating the following mutations: thyA, thyB, uvr-1, dal, spoOA delta 677, citD delta 20 (uvrC), strd. The strain is unable to grow in the absence of thymine, D-alanine, or streptomycin; unable to sporulate; and sensitive to UV radiation. Survival of the attenuated strain was severely restricted in nonpermissive environments, and the organism suffered a five-log decrease in viability within 24 h when dried at room temperature. The strain could be transformed with plasmid DNA using the protoplast transformation system and was transducible with PBS1. The ability of the strain to serve as host for the temperate phages phi 3T and rho 11 and for the plasmid vectors pUB110 and pBD64 was unimpaired.

To count V kappa genes homologous to particular V kappa nucleotide sequences, thereby permitting estimates for the total V kappa repertoire, we hybridized 10 cloned V kappa cDNA sequences to restriction fragments of mouse embryo DNA (Southern blots). Particular probes labeled up to 17 fragments, of which up to 8 were strongly labeled. This indicates that the germline contains sets of related V kappa genes. Only 4 nonoverlapping gene sets of 16-22 genes each were found. Assuming that the probes used are a random sample of the V kappa pool, the pattern repetitions suggest that the germline contains a total of about 5 distinct V kappa gene sets and about 90 V kappa genes. Correlating sets of strongly labeled genes with V kappa groups having similar N-terminal sequences led to an estimate of about 300 germline V kappa genes, while extrapolation from the number of genes in the VK-21 group gave an estimate of 90-140 genes. Since the three independent estimates fell between 90 and 320 genes, the germline V kappa repertoire, at least as expressed in myelomas, probably lies in that range. The V kappa fragment patterns given by three probes did not differ detectably between BALB/c, NZB, and A/J DNA but all three patterns differed in AKR DNA, indicating that there is limited polymorphism within the mouse V kappa locus. Somatic mutation must expand the number of expressed V kappa sequences, since the 8-12 VK-21 genes estimated for BALB/c and for NZB is significantly less than the 22 known NZB VK-21 polypeptides.

In the course of studying the members of the T15 group of VH gene segments, some of which participate in the immune response to phosphorylcholine in the mouse, we identified a VH gene segment that contains three mutations preventing its expression. The mutations are an in-frame stop codon, a 4-base insertion which causes a termination codon to be shifted into the reading frame, and a modification of the recognition elements involved in the joining of VH and D gene segments during variable region formation. This pseudogene, which is 88-96% homologous to the other members of the T15 VH gene group, is probably of relatively recent origin and will presumably be deleted from the VH gene family eventually. We suggest that pseudogenes can only arise in multigene families and that the occurrence of pseudogenes will be a relatively frequent phenomenon in these families. Because the antibody gene families are made up of multiple gene elements, undergo two types of DNA rearrangements during differentiation, and employ several different RNA splicing mechanisms for expression, there are many different ways a particular antibody gene segment may become a pseudogene.

We have transformed Chinese hamster ovary cells with a plasmid containing mouse dihydrofolate reductase (DHFR) cDNA and the Escherichia coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene under the control of the mouse mammary tumor virus and SV40 early promoters, respectively. Selection for the expression of XGPRT using the dominant selection scheme described by Mulligan and Berg yields clones that simultaneously express DHFR. Growth of these cells in progressively increasing concentrations of methotrexate, results in selection of cells that overproduce DHFR and its messenger RNA 250-500 fold. ANalyses of the plasmid DNA sequences in these cells reveal that the increased production of DHFR is due in part to gene amplification (approximately 50-fold) and in part to a selective overproduction of DHFR RNA. Last, the methotrexate-resistant cells contain 50 times more XGPRT RNA and DNA than the initial transformant; this demonstrates the potential for using gene amplification as a means for overproducing the products of genes linked to DHFR cDNA in plasmid vectors.

The gene encoding the common alpha subunit of the four human glycoprotein hormones, chorionic gonadotropin (CG), luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH), has been cloned in a bacteriophage lambda vector. Restriction endonuclease digestion of total human DNA suggests that the common alpha subunit is coded for by a single gene. Three distinct polymorphic hybridization patterns have been observed for this gene in the human population. The cloned gene encompasses a total of 9.4 kilobases (kb) and contains three intervening sequences whose locations have been established by restriction enzyme mapping and by DNA sequencing. One of the intervening sequences is located in the 5' untranslated region, generating a leader sequence that is separated from the rest of the gene by 6.4 kb. The other two intervening sequences are 1.7 and 0.4 kb long and are located within codon number 6, and between codons 67 and 68, respectively. The location of the 5' end of the mature transcript has been established by priming placental mRNA with a restriction fragment obtained from the cloned cDNA. A transcript of similar size for the alpha subunit gene has been detected in both the pituitary, where the gene is expressed for the synthesis of LH, FSH, and TSH, and the placenta, where the gene is expressed for the synthesis of CG. When parts of the 5' untranslated nucleotide sequences of the alpha subunit and the human growth hormone genes are compared a highly homologous region is observed. These otherwise unrelated genes share the common feature that they encode a secreted pituitary polypeptide hormone.

The polyoma virus early region mRNAs synthesized during productive infection of mouse cells have been characterized at the nucleotide level. One- and two-dimensional agarose gel fractionation of nuclease S1-resistant RNA-DNA hybrids was used to establish basic structures. The two splice donors and the two splice acceptors were positioned more precisely by high resolution S1-gel mapping with terminally labeled DNA probes and polyacrylamide gels. The nucleotide sequences across the three splice joints were established by cloning and sequencing partial cDNA copies of the mRNAs. In combination with data on the polyadenylated 3'-end previously published, and the detailed analysis of the capped 5'-ends presented elsewhere, the present data complete the description of a family of differentially spliced mRNAs able to encode the known early region gene products, small-T, middle-T, and large-T proteins.

A goat strain with congenital goiter was studied as a model for human thyroid disoders. These goats were deficient in thyroglobulin (Tg), the precursor protein of the thyroid hormones T3 and T4. RNA coding for Tg (Tg-RNA) was detected in reduced amounts in the goiters and was almost absent from the membranes, where Tg is normally synthesized. This paper describes the preparation and characterization of a goat Tg cDNA plasmid and its use in the study of thyroglobulin gene expression in the goiter. We found that the goiter Tg-RNA is polyadenylated and has the same size as normal 33S goat TG mRNA. Thus, there are no major defects in the mRNA processing. However, in contrast to normal Tg mRNA, the goiter Tg-RNA was not translated into immunoprecipitable Tg subunits when injected into Xenopus oocytes. We conclude therefore that the goiter Tg-RNA has one or more alterations causing a lack of proper translation and/or a decreased cytoplasmic stability.

Biosynthesis of the alpha', alpha, and beta subunits of the soybean 7S storage protein (conglycinin) was studied by in vivo and in vitro experiments. One hour in vivo labeling produced polypeptides of 72 kilodaltons (kD) and 78 kD which, in a subsequent 1-h chase period, gave rise to a heterodisperse band of polypeptides of 76-83 kD, the apparent molecular weights of mature alpha and alpha', respectively. After a 3-h chase period only mature alpha and alpha' were labeled. The pre-alpha' (80 kD) and pre-alpha (78 kD) polypeptides produced by in vitro translation of total seed poly(A)+ RNA did not coelectrophorese with polypeptides labeled in vivo. The mature beta-subunit (53 kD), produced in vivo during the 1-h chase period, apparently was translated as a 50 kD polypeptide in vitro. The data suggest that, in vivo, primary translation products undergo both cotranslational and posttranslational modifications during the formation of mature subunits. cDNA clones complementary to soybean seed poly(A)+ RNA were prepared and subsequently identified by hybrid-release and immunoprecipitation experiments. Clone pGmc 236 (550 base pairs) (bp) was shown to contain sequences complementary to mRNAs for the alpha', alpha, and beta subunits on the basis of hybrid-release experiments, corroborating tryptic fingerprints that demonstrate that these subunits are closely related to each other. Although selected by a single cDNA clone, the mRNAs are not identical because the alpha' mRNA was eluted from the filter-bound cDNAs at a lower temperature than were the alpha beta mRNAs, pGmc 236 hybridized on Northern blots to mRNAs of approximately 2500 nucleotides, sufficient size to code for the alpha' or alpha subunit of the 7S storage protein.

It is difficult to clone directly some regulatory or structural genes on the basis of their functions, because of their obscure properties or the leakiness of their mutants. To overcome this problem, a two-step cloning method with use of phage Mu was developed and applied to cloning of the ompB gene, an obscure regulatory gene for major outer membrane proteins of Escherichia coli. The ompB gene was first inactivated by phage Mu insertion, and the approximately 25-kilobase (kb) EcoRI fragment, which hybridized with phage Mu DNA, was cloned into a plasmid vector, pBR322. This DNA fragment was considered to contain not only a portion of phage Mu DNA but also a portion of the ompB gene DNA. With this DNA as a probe, the wild-type ompB+ strain was found to contain a 12.7-kb EcoRI fragment which hybridized with the probe. In the second step, this 12.7-kb EcoRI fragment was cloned into a lambda phage vector, lambda 569. Lysogenization of an ompB mutant with this phage suppressed OmpB- phenotypes, indicating that the 12.7-kb EcoRI fragment carried the ompB gene. The same 12.7-kb DNA fragment, as well as a 3.8-kb EcoRI-BamHI subfragment, was cloned into pBR322. Both plasmid clones were able to suppress the OmpB- phenotypes upon transformation of an ompB mutant.