Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange.

The Class Bdelloidea of the Phylum Rotifera is the largest metazoan taxon in which males, hermaphrodites, and meiosis are unknown. We conducted a molecular genetic test of this indication that bdelloid rotifers may have evolved without sexual reproduction or genetic exchange. The test is based on the expectation that after millions of years without these processes, genomes will no longer contain pairs of closely similar haplotypes and instead will contain highly divergent descendants of formerly allelic nucleotide sequences. We find that genomes of individual bdelloid rotifers, representing four different species, appear to lack pairs of closely similar sequences and contain representatives of two ancient lineages that began to diverge before the bdelloid radiation many millions of years ago when sexual reproduction and genetic exchange may have ceased.

The Sverdlovsk anthrax outbreak of 1979.

In April and May 1979, an unusual anthrax epidemic occurred in Sverdlovsk, Union of Soviet Socialist Republics. Soviet officials attributed it to consumption of contaminated meat. U.S. agencies attributed it to inhalation of spores accidentally released at a military microbiology facility in the city. Epidemiological data show that most victims worked or lived in a narrow zone extending from the military facility to the southern city limit. Farther south, livestock died of anthrax along the zone's extended axis. The zone paralleled the northerly wind that prevailed shortly before the outbreak. It is concluded that the escape of an aerosol of anthrax pathogen at the military facility caused the outbreak.

Interspecific nucleotide sequence comparisons used to identify regulatory and structural features of the Drosophila hsp82 gene.

We have sequenced the hsp82 heat shock gene and 5'-flanking DNA of four species of Drosophila in order to identify conserved features of possible regulatory significance and to determine the nature and rate of evolutionary change in various domains of the gene. All conserved sequences identified in the 5' non-transcribed region of the hsp82 gene of melanogaster, simulans, pseudoobscura and virilis lie within 150 base-pairs of the RNA initiation site. These include a 34 base-pair imperfect but highly conserved dyad made up of three overlapping copies of the consensus heat shock regulatory sequence dyad made up of three overlapping copies of the consensus heat shock regulatory sequence C-T-N-G-A-A-N-N-T-T-C-N-A-G. Several other highly conserved features are also seen. In pseudoobscura only, the region from -21 to -133 is almost exactly repeated 698 base-pairs upstream. The upstream repeat retains all of the sequences common to the four species in the -21 to -133 interval except for the T-A-T-A motif. One possibility is that this upstream sequence is involved in dosage compensation of the hsp82 gene, which is pseudoobscura is on the X chromosome. In melanogaster, simulans and virilis there is an oppositely oriented, non-heat shock gene 0.6 to 0.8 kb (kb = 10(3) base-pairs) upstream from the hsp82 transcription initiation site. Comparisons of the hsp82 transcription unit from the distantly related species show little or no sequence conservation in the intron and non-translated exon I sequences. In contrast, the coding regions of these species are 90% homologous at the DNA level and 97 to 99% identical at the amino acid level. Half of the amino acid changes have occurred within a 15-amino acid region that lies within an unusually polar domain of hsp82. From the sequence comparisons, we estimate the rate of silent (synonymous) site substitution in Drosophila as 1 X 10(-8)/nucleotide site per year, similar to that for mammals.

On the mode of gene-dosage compensation in Drosophila.

A procedure is described for determining the mode and magnitude of gene-dosage compensation of transformed genes. It involves measurement of the ratio of the activity of a gene inserted at X-linked sites to the activity of the same gene inserted at autosomal sites. Applying the procedure to the Drosophila pseudoobscura Hsp82 gene inserted at ectopic sites in D. melanogaster and taking gene activity as proportional to the amount of transcript per gene copy, we conclude that (1) in both adults and larvae the gene is not compensated at autosomal sites or at a site in beta-heterochromatin at the base of the X chromosome and is fully compensated at euchromatic X-chromosomal sites; (2) inappropriate normalization is responsible for a claim that the gene is compensated at autosomal sites; and (3) the observed compensation operates mainly or entirely by heightened activity of X-linked genes in males, rather than by reduced activity in females.

Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli.

Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.

Genetic interactions of modifier genes and modifiable alleles in Drosophila melanogaster.

We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.

Plasmid screening at high colony density.

A procedure is described for screening bacterial colonies containing recombinant plasmids by nucleic acid hybridization at high density, i.e., at 100 000 colonies per 150 mm diameter plate. Small colonies are established on nitrocellulose filters from which they can be faithfully replicated to additional filters. Chloramphenicol amplification may be carried out in situ before screening. The filters may be kept frozen for long-term storage of colonies which may be further replicated after thawing.