RESUMEN
The distribution of interspersed repetitive DNA sequences in the human genome has been investigated, using a combination of biochemical, cytological, computational, and recombinant DNA approaches. "Low-resolution" biochemical experiments indicate that the general distribution of repetitive sequences in human DNA can be adequately described by models that assume a random spacing, with an average distance of 3 kb. A detailed "high-resolution" map of the repetitive sequence organization along 400 kb of cloned human DNA, including 150 kb of DNA fragments isolated for this study, is consistent with this general distribution pattern. However, a higher frequency of spacing distances greater than 9.5 kb was observed in this genomic DNA sample. While the overall repetitive sequence distribution is best described by models that assume a random distribution, an analysis of the distribution of Alu repetitive sequences appearing in the GenBank sequence database indicates that there are local domains with varying Alu placement densities. In situ hybridization to human metaphase chromosomes indicates that local density domains for Alu placement can be observed cytologically. Centric heterochromatin regions, in particular, are at least 50-fold underrepresented in Alu sequences. The observed distribution for repetitive sequences in human DNA is the expected result for sequences that transpose throughout the genome, with local regions of "preference" or "exclusion" for integration.
Asunto(s)
Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Cromosómico , ADN Recombinante , Humanos , Modelos Genéticos , Hibridación de Ácido NucleicoRESUMEN
By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).
Asunto(s)
Genes Bacterianos , Rhizobiaceae/genética , Simbiosis , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/genética , Prueba de Complementación Genética , Hibridación de Ácido Nucleico , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Rhizobiaceae/genética , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasa EcoRI , Desoxirribonucleasa HindIII , Electroforesis en Gel de Agar , Prueba de Complementación Genética , Mutación , Hibridación de Ácido NucleicoRESUMEN
The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.