Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego.

A gene from Bacillus thuringiensis subsp. san diego that is responsible for a delta-endotoxin active against Colorado potato beetle and some other Coleoptera was sequenced and shown to have surprising regional homology with both lepidopteran and dipteran active delta-endotoxins from other strains of B. thuringiensis. Unlike the lepidopteran active toxins from B. thuringiensis subsp. kurstaki that exist as approx. 130-kDa protoxins and form bipyramidal crystalline inclusions, the coleopteran toxic protein forms a square-shaped crystal composed of an approx. 65-kDa protein. Comparisons of the gene sequences encoding the active portions of these protoxins indicate conservation of N-terminal hydrophilic and hydrophobic regions, and suggest a distant ancestral origin for these insecticidal proteins.

A method for analyzing transcription using permeabilized cells.

Brief exposure of Friend cells to a buffered hypotonic solution containing 1% Tween 80 caused permeabilization and allowed incorporation of [3H]UTP into RNA. The incorporation was inhibited 85-97% by 20 micrograms/ml actinomycin D and the reaction product was completely hydrolyzed by 0.1 M KOH. UMP incorporation was nearly linear for 60 min at 23 degrees C; however, at 37 degrees C it ceased after 15-20 min of rapid incorporation. The inhibition of UMP incorporation by 2 micrograms/ml alpha-amanitin was much greater at 23 degrees C than at 37 degrees C. The molecular weight of the RNA synthesized in permeabilized cells is broadly distributed with about 83% larger than 18 S. In vitro transcription of the mouse beta-major globin gene was studied by hybridizing 32P-labeled nascent RNA to filter-bound DNA sequences representing this gene and its flanking regions. After induction by hexamethylene-bisacetamide, Friend cells exhibited more than fivefold increases in the rate of transcription for the beta-major globin gene as compared to the uninduced control cells. Induction also caused an increase in the transcription rate of the 3'-flanking region located downstream from the poly(A) addition site. Thus, the primary transcription unit of beta-major globin gene is essentially the same in permeabilized cells as that previously reported for nuclei isolated from the same cell line. In addition, permeabilized cells actively initiate RNA synthesis as determined by the incorporation of a thiol group at the 5' initiating nucleotide, when synthesis was in the presence of [gamma-S]-labeled nucleoside triphosphates. Permeabilized cells are about 7-11 times more active than isolated nuclei in the synthesis of both in vitro-initiated and total RNA.

The analysis of some new Drosophila repetitive DNA sequences isolated and cloned from two-dimensional agarose gels.

Drosophila melanogaster DNA (Dm) was sequentially cleaved by BamHI and EcoRI and separated by two-dimensional gel electrophoresis. Six different prominent bands, which are derived primarily from the cleavage of long sequences that are repeated 20-100 times per genome, were recovered from the gel and cloned in pBR322. Hybridization and restriction analysis of the cloned Dm segments showed that three of these bands are mainly derived from the ribosomal and histone gene repeating units. Segments cloned from the other three bands are not homologous to any known repeating elements that we have tested. They represent long repetitive sequences of moderate multiplicity that appear not to have been hitherto described. These segments have been restriction-mapped and hybridized to cDNA prepared from poly(A)RNA from adult flies. While two minority segments did hybridize to this probe, the majority failed to hybridize. The arrangement of genomic sequences homologous to each plasmid was tested by restriction analysis and Southern hybridization. The results indicate that the repetitive element is largely conserved intact although occupying numerous different positions in the genome. The DNAs from four different strains of D. melanogaster and two of D. simulans produced restriction patterns having some segment lengths in common and some showing clear differences, a fact that indicates that these sequences can move about to occupy different genomic locations in different strains.

The influence of agarose--DNA affinity on the electrophoretic separation of DNA fragments in agarose gels.

The effects of DNA concentration, buffer composition, added "carrier" DNA, and chemical modification of agarose on the electrophoretic separation of DNA restriction fragments in agarose gels were tested. Electrophoretic zones of migrating DNA were found to broaden by trailing as sample load was decreased, and this effect was found to be more pronounced for species of higher molecular weight. As DNA sample load was increased, DNA fragments were found to move faster in the direction of electrophoresis (front forward). Sharp, well-resolved electrophoretic zones were obtained at very low DNA loads only when a high-salt, high-pH, high-EDTA buffer was employed or when "carrier DNA" having a broad and uniform molecular weight distribution was included in the sample. Moreover, DNA in high concentration was found to displace DNA in low concentration from a given gel region. Unmodified agaroses were found to differ only slightly in their effectiveness in retarding DNA fragments at a given agarose concentration. However, hydroxyethylated agarose was much more effective in retarding DNA, at a given gel concentration, than the unmodified agaroses tested. These results show that it is useful to consider the agarose gel matrix as possessing the properties of both a molecular sieve and a chromatographic adsorbent when designing electrophoretic separation techniques for DNA. A model for these separations which includes the effects of DNA-agarose interaction and molecular sieving is discussed.

Genetic control of glycolysis in human erythrocytes.

We have studied heritability of the concentration of each glycolytic intermediate and adenine nucleotide in the cytosol of human erythrocytes obtained from a random sample of apparently healthy young individuals. Preliminary to analysis of heritability, each trait was statistically described and the effects attributable to variation in measured concomitants were removed by regression. Heritability was estimated using the family-set method. This method removes covariances between the index case, sibling and first cousin, due to those environmental determinants of the phenotypic values that are shared with a matched, unrelated control member of the family set. It also removes covariances due to environments that are shared by siblings and first cousins. Heritability was estimated by employing the fact that the variance of differences between first cousins minus the variance of differences between full siblings estimates three-fourths of the additive genetic variance. The heritability estimates for G6Pdagger, F6P, ATP and some other metabolite concentrations are high and significantly greater than zero. The heritabilities of G6P and F6P are likely attributable to genetic variation in the in vivo activity of HK and/or PFK, because the concentrations of these metabolites are tightly controlled by the two regulatory enzymes. Statistically significant heritability estimates for HK and PFK mass action ratios strongly suggest genes are responsible for a portion of the quantitative variation in these enzyme activities. Since HK and PFK regulate glycolysis and the production of ATP, genetic variation in their activities might be causally related to the heritability of ATP concentration.