Genetic basis of the neurovirulence of pseudorabies virus.

Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (US) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessary for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-]) revealed the following. None of the recombinant plaque isolates that were either TK- or which had a deletion in the US was virulent. Not all recombinant plaque isolates which were both TK+ and had an intact US were virulent. These results indicate that both thymidine kinase activity and an intact US were necessary but not sufficient for the expression of virulence. Marker rescue experiments involving cotransfection of the Bartha strain DNA and a restriction fragment spanning the region of the genome that was missing from the Bartha strain resulted in the isolation of virions to which an intact US had been restored. These virions were not virulent but had an improved ability to replicate in the brains of chicks compared with that of the parental nonrescued Bartha strain. Our results show that genes in the US region, which are missing from the Bartha strain, were necessary for virulence but that this strain was also defective in other genes required for the expression of virulence. Thus, the virulence of pseudorabies virus, as measured by intracerebral inoculation into chicks, appears to be controlled multigenically.     

Nitrate reductase: an improved assay method for phytoplankton

A new assay for measuring the activity of nitrate reductasein phytoplankton, based upon the permeability of cells treatedwith toluene to substrates and products, is described. The methodis simple and, since the reaction is carried out directly ona glass fiber filter, can be easily performed in the field oron shipboard. In comparison with previous methods, this techniquegave higher absolute amounts of NO^–₂ formed per unit tuneand higher enzymatic activities per sample volume when testedwith axenic algal cultures and with natural phytoplankton populationsfrom Lake Kinneret, the River Jordan and the Eastern Mediterranean.     

Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.     

Genetic diversity and environmental associations of wild barley,Hordeum spontaneum (Poaceae), in Iran

Genetic diversity and structure of populations of the wild progenitor of barleyHordeum spontaneum in Iran was studied by electrophoretically discernible allozymic variation in proteins encoded by 30 gene loci in 509 individuals representing 13 populations of wild barley. The results indicate that: a)Hordeum spontaneum in Iran is extremely rich genetically but, because of predominant self-pollination, the variation is carried primarily by different homozygotes in the population. Thus, genetic indices of polymorphismP-1% = 0.375, range = 0.267–0.500, and of genetic diversity,He = 0.134, range = 0.069–0.198, are very high. b) Genetic differentiation of populations includes clinal, regional and local patterns, sometimes displaying sharp geographic differentiation over short distances. The average relative differentiation among populations isGst = 0.28, range = 0.02–0.61. c) A substantial portion of the patterns of allozyme variation in the wild gene pool is significanctly correlated with the environment and is predictable ecologically, chiefly by combinations of temperature and humidity variables. d) The natural populations studied, on the average, are more variable than two composite crosses, and more variable than indigenous land races of cultivated barely,Hordeum vulgare, in Iran. — The spatial patterns and environmental correlates and predictors of genetic variation ofH. spontaneum in Iran indicate that genetic variation in wild barley populations is not only rich but also at least partly adaptive. Therefore, a much fuller exploitation of these genetic resources by breeding for disease resistance and economically important agronomic traits is warranted.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.     

Transposable elements in mendelian populations. I. A theory

Transposable elements are DNA sequences, found throughout eukaryotes, that transpose replicatively and cause numerous genetic and developmental effects on their hosts. A model of the evolution of transposable elements in Mendelian populations is proposed. From its analysis, formulas for the mean copy number and frequency spectrum are obtained.     

Insulin as a surface marker on isolated cells from rat pancreatic islets

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.