Molecular cloning, sequencing, and expression of two late proteins of bacteriophage MB78

Bacteriophage MB78, a virulent phage of Salmonella typhimurium, does not allow other phages, such as P22 and 9NA, to grow in its presence. A detailed physical map of this phage has been constructed in our laboratory. In an ongoing effort to understand the genetics of this interesting phage, various genes were characterized. Here, we report cloning, sequencing, and expression of two late proteins, coded in a SalI-HindIII fragment (SH9), by using the minicell expression system. Further, we performed a kinetic study of phage proteins by infection the host LT2 cells and compared the proteins produced, with proteins obtained by the minicell expression system. Both sets of proteins run exactly parallel and migrated as 14- and 15-kDa proteins on a polyacrylamide gel. The synthesis of these two proteins started 15 min after infection with MB78 and was prominent after 45 min. One of the proteins exhibited 57% homology to the structural protein of mycobacteriophage L5.     

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.     

Is the injection of DNA enough to cause bacteriophage P22-induced changes in the cellular transport process of Salmonella typhimurium?

It was demonstrated earlier in this laboratory that phage P22 induces a transient depression in the cellular transport processes of the host Salmonella typhimurium immediately after infection and that an effective injection process is enough to cause the depression. By using defective phage particles that contain host DNA instead of phage DNA for infection, it has been demonstrated that the injection of phage-specific DNA is essential for this. The defective particles adsorbed to the host and injected their DNA, but the cellular transport processes of the host were not altered. Thus, the injection of host DNA by the phage fails to affect the transport process. Insensitivity of the phage DNA-induced depression in transport to chloramphenicol rules out the involvement of newly synthesized protein in this change and indirectly suggests the possible role of phage DNA-associated internal proteins of P22.     

Milk-Clotting Enzymes From Microorganisms

A total of 230 cultures of fungi and 43 cultures of bacteria, isolated from such sources as soil, butter, and milk, were screened for their milk-clotting activity. The fungi were cultivated on semisolid media, and the bacteria were grown in milk media in shake culture. Phytic acid, added as calcium phytate, was found to stimulate production of the enzyme in most of the bacterial isolates. Proteolytic activity was invariably found to be associated with the milk-clotting enzyme in bacterial isolates. There was considerable variation in the ratio of the two enzymes from strain to strain.     

Genetic Variation within Native Populations of Endemic Silkmoth Antheraea assamensis (Helfer) from Northeast India Indicates Need for In Situ Conservation

A. assamensis is a phytophagous Lepidoptera from Northeast India reared on host trees of Lauraceae family for its characteristic cocoon silk. Source of these cocoons are domesticated farm stocks that crash frequently and/or wild insect populations that provide new cultures. The need to reduce dependence on wild populations for cocoons necessitates assessment of genetic diversity in cultivated and wild populations. Molecular markers based on PCR of Inter-simple sequence repeats (ISSR) and simple sequence repeats (SSR) were used with four populations of wild insects and eleven populations of cultivated insects. Wild populations had high genetic diversity estimates (H_i = 0.25; H_S = 0.28; H_E = 0.42) and at least one population contained private alleles. Both marker systems indicated that genetic variability within populations examined was significantly high. Among cultivated populations, insects of the Upper Assam region (H_i = 0.19; H_S = 0.18; H_E = 0) were genetically distinct (F _ST = 0.38 with both marker systems) from insects of Lower Assam (H_i = 0.24; H_S = 0.25; H_E = 0.3). Sequencing of polymorphic amplicons suggested transposition as a mechanism for maintaining genomic diversity. Implications for conservation of native populations in the wild and preserving in-farm diversity are discussed.     

Juvenoid-induced effects on the growth and differentiation of testis in the rice moth, Corcyra cephalonica

The juvenoid hydroprene or altozar (ZR-0512), when applied to larvae and pupae of Corcyra cephalonica at doses of 100 μg and 10 μg per individual, produced abnormalities in the growth and differentiation of imaginal testicular structures. In all the resultant larval-imaginal intermediates each testis contained a compact mass of cysts at various developmental stages with a comparatively higher number of spermatid cysts denoting a potentiality for increased fertility. Delayed differentiation of spermatozoa may also be inferred from this finding. On rare occasions the two larval testes failed to become enclosed in a single sac. Conversely, in the adultoids obtained from pupal treatment, the production of a normal quantity of testicular cysts of different kinds was inhibited. Moreover, malformed and sometimes degenerating spermatozoan cysts were formed. In a few extreme cases differentiation of even the spermatid cysts had been almost entirely prevented. Spermatogonia and the degenerating cell masses were the chief components of the testis of such adultoids. All observations indicated a sort of male sterility induced by the juvenoid when applied to pupae, especially at 12 hours age. The effect of the juvenoid on spermatogenesis could not be shown to be direct.