Occurrence of different types of infectious hematopoietic necrosis virus in fish

The virion protein patterns of 71 isolates of infectious hematopoietic necrosis virus (IHNV) from the Pacific Northwest were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]-methionine-labeled virus. This analysis led to the classification of these virus isolates into four or more types. Type 1 virus was characterized by a nucleocapsid protein with an approximate molecular weight of 40,500. Type 2 and type 3 viruses have nucleocapsid proteins with molecular weights of 42,800 and 43,250, respectively. Type 2 virus was responsible for the recent epizootics of IHNV among fish in the lower Columbia River. The California IHNV isolates were type 3 with the exception of some of those isolated from fish at the Coleman Hatchery on the Sacramento River. These Coleman Hatchery isolates belonged to a type 4 virus group characterized by a larger glycoprotein of approximately 70,000 molecular weight. All other viruses examined had glycoproteins of 67,000 molecular weight. The "type 5" virus isolates were grouped together because they were not sufficiently distinct to warrant classification into a separate type. These findings have been useful in determining that a particular virus type is characteristic for a geographic area and will infect many different salmonid species in that area and the same type isolated from parental fish is responsible for the subsequent outbreak of the diseases in progeny.     

Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression.

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.     

Genomic analysis II: isolation of high molecular weight heteroduplex DNA following differential methylase protection and Formamide-PERT hybridization.

Understanding the nature of DNA sequence differences among individuals is important to the understanding of fundamental questions in biology. To analyze such differences in complex genomes new approaches must be developed. We report two new techniques which aid in this effort. First, we have developed a modification of the Phenol Emulsion Reassociation Technique (PERT) that allows hybridization of long (20 kb and longer) single copy heteroduplex DNA fragments from human genomic DNAs. Secondly, by using a differential methylase protection technique we have shown that double methylase resistant heteroduplex DNA molecules can be size fractionated away from reannealed single methylase resistant homoduplex DNA molecules. These methods will be useful in obtaining DNA from chromosomal subregions linked to the inheritance of a specific trait or condition as described in the preceding paper and could also be used to create a map of the chromosomal subregion which includes the gene for the trait.     

Assessment of binding of L-cystine and L-lysine by rat renal brush-border membranes

Cystine and lysine bind to isolated rat renal brush-border vesicles. Three methods to determine the extent of amino acid binding to the membranes have been compared, one relying on the osmotic reactivity of the vesicle, a second by trichloroacetic acid precipitation of membrane-bound material and a third by initial rate analysis. For cystine, all methods yield comparable results at early time points, indicating the trichloroacetic acid method is a simple and valuable tool for binding estimation under initial-rate or near initial-rate conditions. For lysine, initial rate analysis and osmotic perturbation are the methods of choice since lysine co-precipitates with trichloroacetic acid.     

Regulation of renal sulfoglycolipid biosynthesis

The in vitro activity of the renal galactolipid sulfotransferase and the level of sulfated glycolipids in the rat kidney have been correlated as a function of age. The galactolipid sulfotransferase was found to be greatly reduced in the young as compared with the adult animal. The relatively minor changes in the sulfated glycolipid content of the kidney with age suggests that an increase in sulfoglycolipid turnover occurs during growth. An inhibitory activity was detected in the homogenate supernate of the young animal capable of reducing the in vitro sulfotransferase activity of the adult. Assay of the human renal galactolipid sulfotransferase showed that this enzyme activity is deleted in samples of the blastematous form of Wilm's renal tumor. The results suggest that the rate of synthesis of renal sulfoglycolipids may prove a marker of renal development, perhaps by post translational regulation.     

Kinetic studies of cellodextrins hydrolyses by exocellulase from Trichoderma reesei

The kinetics of the hydrolyses of cellotriose and of cellotetraose by cellobiohydrolase were studied using a convenient integral technique. Reaction mechanisms and mathematical models were postulated to describe the reactions. The end-products of the reaction were found to be inhibitory toward hydrolysis in a competitive mode. Hydrolysis of cellotetraose produces cellobiose and hydrolysis of cellotriose produces cellobiose and glucose. Both sugars inhibit the enzyme with cellobiose being a stronger inhibitor.     

Uracil's uncoupling of the decarboxylation of alpha-ketoglutarate in the thymine 7-hydroxylase reaction of Neurospora crassa

Highly purified preparations of thymine 7-hydroxylase from Neurospora crassa catalyzed the decarboxylation of alpha-ketoglutarate but yielded no hydroxylated product when uracil was substituted for thymine in the standard incubation mixture. Although the uracil-dependent decarboxylation was much slower than the coupled reaction, both reactions were similar with respect to the requirement for molecular oxygen, the stoichiometric formation of succinate, and the stimulations effected by Fe2+, ascorbate, and catalase. That the same enzyme catalyzed both reactions was indicated by the parallel loss of the uracil- and thymine-dependent activities upon heat denaturation, their copurification, and the lower level of both activities in a mutant strain deficient in the 7-hydroxylase. These data are consonant with molecular oxygen initially attacking alpha-ketoglutarate in the thymine 7-hydroxylase reaction.     

Evolutionary conservatism in arrangement of genetic material

The diploid number of the Rhesus macaque, Macaca mulatta, is 42. All chromosomes are biarmed and all constitutive heterochromatins are centromeric. The diploid number of the African Green monkey, Cercopithecus aethiops, is 60. Again all chromosomes are biarmed, but seven pairs possess very short second arms which are heterochromatic. The heterochromatins of remaining chromosomes are centromeric. Using G-banding and deleting the heterochromatic short arms, the chromosomes of the African Green monkey can be artificially fused to reconstruct a karyotype of the Rhesus with only one pair of unmatched small metacentrics. In addition to the Robertsonian type of translocations, several sets of centromere-telomere translocations were found. The latter type of translocation reduced three arms into two. Thus the fundamental number can be changed by two mechanisms: growing extra heterochromatic arms and the centromere-telomere fusions.     

Pure Culture Studies of Erwinia carotovora with 3,5-Diiodo-4-Hydroxybenzonitrile

Interactions of ioxynil (3,5-diiodo-4-hydroxybenzonitrile) with a pure culture of Erwinia carotovora grown in a glucose-simple salts medium were studied. Growth of E. carotovora was inhibited by ioxynil and, to a lesser extent, by its acid form at 25 and 50 mug/ml. Growth was not inhibited by the amide or ester forms of ioxynil or p-hydroxybenzonitrile at the same concentrations. E. carotovora could be trained to grow in 50 mug or higher concentrations of ioxynil per ml by serial transfers of the organism through increasing ioxynil concentrations. No degradation or detoxification of ioxynil was detected. Toxicity tests indicated that, in the adapted culture, cell-free supernatant fluid remained toxic to a nonadapted culture. Adaptation of E. carotovora resulted in a lengthened lag phase, a decreased growth rate, and very few adverse effects on the total population. The adapted resistant culture retained this characteristic only when ioxynil was present. Adaptation was demonstrated to be a physiological variation, not a selection of a mutant or of preexisting resistant cells. Ioxynil slightly stimulated the respiration rate of E. carotovora and moderately inhibited that of an adapted culture. Because the respiration rate of an adapted culture in the absence of ioxynil surpassed that of a parent culture still in the presence of ioxynil, a competition of two alternate routes of electron transport is implied. These data support the conclusion that an alternate growth mechanism is involved in the adaptation mechanism.