Localization of mouse type 2 Alu sequence in schistosomes

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8- week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.

DNA hybridization analysis of thePseudomonas aeruginosaelastase gene [lasB) from different clinical isolates

Pseudomonas aeruginosa produces several extracellular virulence factors including elastase (which is encoded by lasB). Recently, we examined several clinical isolates of P. aeruginosa for the production of toxin A, elastase, exoenzyme S, and phospholipase C. Although the majority of the isolates produced a high level of elastase, a few isolates produced either very low or no detectable elastase. In this study, we tried to determine the presence of restriction site heterogeneity within lasB from these isolates and the possible correlation between such heterogeneity and the observed variation in elastase production. Chromosomal DNA from the isolates was digested with different restriction enzymes and examined by Southern blot hybridization experiments using two lasB probes. One lasB probe covers 636 bp of lasB structural gene while the other covers 240 bp of the lasB upstream region. Chromosomal DNA from P. aeruginosa PAO1 and PA103 was used as controls. Results indicate that chromosomal DNA from all isolates hybridized to both lasB probes. Depending on the restriction enzyme used for DNA digestion, lasB from 3 to 12% of the isolates showed different patterns of hybridization with the lasB structural gene probe. However, no difference in the hybridization pattern was seen with the lasB upstream probe. With the exception of one isolate, hybridization of genomic DNA from different isolates (with both probes) produced a single hybridization band. In that isolate, an additional hybridization band was detected. Immunoblotting experiments confirmed that elastase protein is not produced by 6 out of 67 isolates. However, lasB from four of these elastase-deficient strains showed no difference in the hybridization pattern with either lasB probe. These results suggest that (i) lasB is present as a single copy in all but one isolate; (ii) with the exception of one, the lasB upstream region from different P. aeruginosa isolates contains no restriction site polymorphism; (iii) the observed heterogeneity within lasB structural genes is limited; and (iv) variations in the hybridization patterns of lasB from different isolates do not correlate with the differences between these isolates in elastase production.Key words: Pseudomonas aeruginosa, clinical isolates, DNA hybridization, elastase, lasB.

Gene disruption by transformation in Neurospora crassa

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.

Gene disruption by transformation in Neurospora crassa.

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.