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The spectrum of mutations induced upon in vivo replication of an M13 genome containing a site-specifically located propanodeoxyguanosine (PdG) adduct was determined. PdG was used as a model for the major deoxyguanosine adduct produced on reaction of DNA with the endogenous genotoxin malondialdehyde. PdG was introduced at position 6256 of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into an 8-base gap in the (-)-strand of duplex M13MB102. Replication of the adducted strand was maximized by incorporation of uracil into the unadducted (+)-strand. Following replication of dG-containing and PdG-containing M13MB102 genomes in Escherichia coli JM105, frameshift mutations were detected as phenotypic changes in the lacZ alpha marker gene. Base pair substitutions were detected by differential hybridization using 32P-labeled 13-mers bearing different bases opposite position 6256. Neither frameshift nor base pair substitution mutations were detected following replication of PdG-adducted genomes in non-SOS-induced JM105. However, PdG-->T transversions and PdG-->A transitions were detected following transformation of PdG-adducted M13MB102 into SOS-induced JM105. Both types of mutations were detected at comparable frequencies, and the total mutation frequency was approximately 2%. The results indicate that PdG is an efficient premutagenic lesion in E. coli strains in which the SOS response is induced.
In previous studies, a mutant (cp-RSV) of the RSV A2 strain derived from 52 serial cold passages in bovine embryonic tissue culture was highly attenuated in seropositive adults and children but caused upper respiratory tract disease in seronegative infants. We investigated the genetic basis for this attenuation phenotype by comparing the complete genomic RNA sequence of this virus with the published sequence of strain A2 as well as with that of its unattenuated wild-type parent (HEK-7) virus. RNA was extracted from virions grown in tissue culture, reverse transcribed, amplified by the polymerase chain reaction (PCR), cloned, and sequenced. Changes from the published A2 wild-type sequence were confirmed on independently derived cDNA clones and by direct sequencing of PCR fragments. The HEK-7 parent virus was then analyzed at these positions by direct sequencing of PCR fragments. Fifteen nucleotide differences between the published A2 wild-type virus and cp-RSV were found. None appeared to involve cis-acting RNA sequences. Of the 15 nucleotide differences, only 1 occurred outside a translational open reading frame (ORF), and 2 which did occur within ORFs were silent at the amino acid level. The remaining 12 nucleotide differences encoded amino acid changes. All 3 of the mutations which were silent at the amino acid level, and 8 of the 12 which resulted in amino acid differences, were also present in the HEK-7 parent virus and therefore were not changes acquired during the cold passages. Thus, the remaining 4 nucleotide differences and the attendant 4 amino acid changes are associated with the attenuation phenotype of the cp-RSV. Two of the changes occur in the F gene and two in the L gene. These results confirm the previously described RSV genomic sequence, provide the first sequence of a live attenuated RSV vaccine strain, provide the first sequence of an RSV strain which has been evaluated in chimpanzees and humans, and indicate that attenuation in humans of a pneumovirus can be associated with a relatively small number of nucleotide and amino acid changes.
In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.
The translocation of the 325 leftmost bp of the genome of pseudorabies virus (PrV) to the internal junction between the L and S components confers upon the virus a growth advantage relative to wild-type PrV in chicken embryo fibroblasts (CEFs) and chickens and a growth disadvantage in rabbit kidney (RK) cells and mice. To clarify the molecular basis for the species-specific growth characteristics of the translocation mutants, we have compared several parameters of the virus growth cycle in CEFs and RK cells infected with wild-type PrV and with translocation mutants. The salient findings are as follows. (i) The synthesis of early-late and late proteins is not as effective in CEFs as it is in RK cells, and these proteins, in particular, the major capsid proteins, accumulate less abundantly in CEFs than in RK cells. (ii) Cleavage of concatemeric DNA to genome-size molecules is also not as effective in CEFs as it is in RK cells. (iii) The internal junction present in translocation mutants is a functional cleavage site. (iv) In RK cells, translocation mutants are hypercleaved and a significant proportion of the total viral DNA is cleaved into subgenomic fragments. (v) In CEFs infected with translocation mutants, subgenomic fragments also accumulate but most of the viral DNA remains in concatemeric form. A model which postulates that the cell-specific growth advantage or disadvantage of the translocation mutants is related to the presence of a second cleavage site within their genomes and is affected by the efficiency of cleavage of concatemeric DNA in particular infected cell types is presented. The significance of these findings as they relate to the evolution of herpesviruses with class 2- and class 3-like genomes is discussed.
A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.
The creation of neural circuits depends on the formation of synapses between specific sets of neurons. Little is known, however, of the molecular mechanisms governing synaptic choice. A mutation in the unc-4 gene alters the pattern of synaptic input to one class of motor neurons in the Caenorhabditis elegans ventral nerve cord. In unc-4(e120), the presynaptic partners of VA motor neurons are replaced with interneurons appropriate to motor neurons of the VB class. This change in neural specificity is not accompanied by any detectable effects on neuronal morphology or process extension. We show that the absence of a functional unc-4 gene product accounts for the mutant phenotype. The unc-4 gene encodes a homeodomain protein and thus is likely to function as a transcription factor. The limited effect of the unc-4 null mutation on cell fate may mean that unc-4 regulates the expression of a small number of target genes and that the products of these genes are directly involved in the choice of synaptic partners.