DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L.) larvae.

Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. To test the assays, neonate larvae were fed droplets containing a known concentration of L. dispar nuclear polyhedrosis virus and observed for up to 10 days to determine the percentage of infected larvae. The average percent mortalities were 88.0, 60.7, 26.0, and 5.3% for larvae fed droplets containing 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) polyhedral inclusion bodies (PIBs) per ml, respectively. Other larvae treated with the same virus concentrations were frozen at 2, 4, and 6 days postinoculation and examined by the hybridization techniques. The average percentage of slot blots containing viral DNA equaled 81.0, 58.0, 18.0, and 6.0% for larvae blotted 4 days after treatment with 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) PIBs per ml, respectively, and 89.9, 52.1, 26.6, and 6.0%, respectively at 6 days postinoculation. Thus, the hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.     

Characterization of Gypsy Moth (Lymantria dispar) Nuclear Polyhedrosis Virus

Characterization of the proteins and nucleic acid of the gypsy moth nuclear polyhedrosis virus isolated in Ithaca, N.Y. (LdNPV-IT) is presented. A total of 29 viral structural proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis when the virus was isolated in the absence of alkaline protease activity. Fourteen surface envelope viral proteins were identified by lactoperoxidase iodination. Eleven proteins were associated with nucleocapsids prepared by Nonidet P-40 detergent treatment. Distinct alterations of viral proteins were documented when virions were purified in the presence of occlusion body-associated alkaline protease(s). Restriction enzyme digests of viral DNA indicated that this isolate was composed of a large number of genetic variants. On the basis of the major molar fragments resulting from EcoRI, BamHI, BglII, and HindIII digests, the molecular weight of the LdNPV genome was approximately 88 × 10⁶.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Field evaluation of a DNA hybridization assay for nuclear polyhedrosis virus in gypsy moth (Lepidoptera: Lymantriidae) larvae.

DNA hybridization assays were used to detect the presence of viral DNA in gypsy moth (Lymantria dispar L.) larvae collected weekly from high density populations or reared from field-collected egg masses. DNA was extracted from larvae, bound to nitrocellulose filters, and hybridized with digoxigenin-labeled L. dispar NPV (LdNPV) DNA probes. The virus incidence determined from DNA hybridization assays was compared with that determined with conventional microscopic examination of larvae for polyhedral inclusion bodies. Among neonates reared from field-collected egg masses, average mortality from LdNPV (15.4%) within 10 d after hatch was not significantly different from the percentage of extracts containing LdNPV DNA (14.8%) found among larvae frozen 5 d after hatch before any mortality occurred. Field-collected larvae were split into two groups: half were frozen immediately and probed for LdNPV DNA and the other half were reared on artificial diet. The proportion containing LdNPV DNA closely approximated the proportion that died within 6 d of collection, but the proportion that died within 13 d of collection was underestimated.     

Physical Maps of Autographa californica and Rachiplusia ou Nuclear Polyhedrosis Virus Recombinants

TN-368 cells were infected simultaneously with the closely related Autographa california (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly cloned plaques, 7 were AcMNPV and RoMNPV recombinants, 5 were RoMNPV, and 88 were AcMNPV. The recombinants contained DNA sequences derived from both parental genomes. By comparing the restriction cleavage patterns of parental and recombinant DNAs, the crossover sites were mapped. A single double crossover was detected in each of the seven recombinant genomes. In addition, six of the seven recombinants revealed a crossover site mapping between 78 and 89% of the genome. The structural polypeptides of the seven recombinants and two parental viruses were analyzed by polyacrylamide gel electrophoresis, and their polyhedrins were identified by tryptic peptide mapping. An analysis of the segregation of three enveloped nucleocapsid proteins and of the polyhedrins among the recombinants located the DNA sequences coding for AcMNPV structural polypeptides with molecular weights of 37,000 (a capsid polypeptide), 56,000, and 90,000 and the RoMNPV structural polypeptides with molecular weights of 36,000 (a capsid polypeptide), 56,000, and 91,000. The AcMNPV and RoMNPV polypeptides of molecular weights 37,000 and 36,000, respectively, mapped within 78 to 89% or 1 to 29%, the polypeptides of molecular weights 55,000 and 56,000 mapped within 78 to 29%, and the polypeptides of molecular weights 90,000 and 91,000 mapped within 19 to 56% of the genome. The region of the parental DNAs that codes for polyhedrin was located within 70 to 89% of the genome.     

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.