Two fragments cloned from purified human satellite III DNA do not cross-react with each other. One fragment, for which a partial sequence is reported, hybridises to satellite II as well as III and is shown to originate on chromosome 1. The other cloned fragment originates from the Y chromosome. This fragment has undergone considerable changes in size when cloned in lambda gt WES lambda B. Human satellite III is shown to consist of a number of non-cross-reacting sequences which nevertheless are related by the presence of closely spaced Hin F1 sites.

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A cosmid library was prepared from a partial BamHJ digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons TnSOl and Tn2l. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADPribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.Bacillus sphaericus is an aerobic spore-forming Bacillus species, several strains of which are pathogenic for mosquito larvae (13). B. sphaericus SSII-1 was isolated in 1973 from infected mosquito larvae collected in India (36). This strain was considerably more toxic than the other B. sphaericus strains known at that time. Early studies indicated that the toxin was retained within or attached to the bacterial cells and that the toxic activity was markedly unstable. Storage of cells under refrigeration or heating at 80°C resulted in the loss of activity. Toxin production occurred predominantly in the vegetative phase of growth before the onset of sporulation (27,29).Further studies on B. sphaericus SSII-1 declined rapidly with the discovery of strains, such as 1593 (36a), 2362 (40), and 2297 (41), which had higher toxicities. In contrast to strain SSII-1, these strains develop relatively stable, high toxicity at the onset of sporulation (7). To date, two types of toxin have been characterized in the highly toxic strains: a pair of proteins of 51.4 and 41.9 kDa associated with the parasporal crystal (3,5,19) and a toxin of 110 kDa related to a 125-kDa surface layer protein (5, 6). Genes encoding the 51.4-and 41.9-kDa proteins were shown to be absent from strain SSII-i (4). In the same study, Baumann et al. showed that sequences inserted in their group C clones were also absent from strain SSII-1. The group C clones were later shown to contain sequences corresponding to the gene encoding the 125-kDa protein (6). Davidson (12) Construction of the cosmid library. To prepare total genomic DNA from B. sphaericus SSII-1, we lysed cells in the presence of 1% sodium dodecyl sulfate and 0.1 mg of proteinase K per ml. DNA was purified from the lysate by the method of Ausubel et al. (2). In brief, hexadecyltrimethyl ammonium bromide was added, and the mixture was incubated at 65°C for 20 min prior to extraction with chloroformisoamyl alcohol. DNA was precipitated with ethanol from the aqueous phase and purified by banding on a cesium chloride gradient coqtaining ethidium bromide. DNA was partially digest...

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Sucl+ was originally identified as a DNA sequence that, at high copy number, rescued Schizosaccharomyces pombe strains carrying certain temperature-sensitive alleles of the cdc2 cell cycle control gene. We determined the nucleotide sequence of a 1,083-base-pair Sucl+ DNA fragment and Si mapped its 866-nucleotide RNA transcript. The protein-coding sequence of the gene is interrupted by two intervening sequences of 115 and 51 base pairs. The predicted translational product of the gene is a protein of 13 kilodaltons. A chromosomal gene disruption of Sucl+ was constructed in a diploid S. pombe strain. Germinating spores carrying a null allele of the gene were capable of very limited cell division, following which many cells became highly elongated. The Sucl+ gene was also strongly overexpressed under the control of a heterologous S. pombe promoter. Overexpression of Sucl+ is not lethal but causes a division delay such that cells are approximately twice the normal length at division. These data suggest that Sucl+ encodes a protein which plays a direct role in the cell division cycle of S. pombe.Among the many genes required for completion of the cell division cycle in the fission yeast Schizosaccharomyces pombe, the cdc2+ gene is of particular interest because it plays a role in both the G, and the G2 phases of the cell cycle. The activity of the gene is required in G, before DNA replication and in G2 before initiation of mitosis (24). There is also physiological and genetic evidence that cdc2+ is not only required in G, and G2 but acts at the rate-limiting step which controls the rate of progression into the S phase and nuclear division (25).The gene product of cdc2+ shares 62% sequence homology with the product of the CDC28 cell cycle "start" gene of the distantly related budding yeast Saccharomyces cerevisiae (15,18). Furthermore, the CDC28 gene can rescue cdc2(Ts) mutants of S. pombe (1), and the cdc2+ gene can rescue cdc28(Ts) strains of S. cerevisiae (7). Both genes encode products which have protein kinase activity (26, 30).The cdc2+ gene was physically isolated from a gene bank of S. pombe DNA by transformation and rescue of a temperature-sensitive cdc2-33 S. pombe strain (1). During screening for DNA sequences which restored a cdc+ phenotype to a cdc2-33 strain, not only cdc2+ but also a second, previously unidentified gene known as Sucl+ was isolated (14). The Sucl + gene, carried on a multicopy number vector pDB248 (2), allowed a cdc2-33 strain to propagate at a nonpermissive temperature. This effect was found to be specific for certain alleles of cdc2. Of the temperaturesensitive alleles tested, cdc2-33, cdc2-56, and cdc2-L7 were rescued by Sucl , whereas cdc2-M35, cdc2-M63, cdc2-M26, and cdc2-M55 were not rescued (14).It has been suggested that the Sucl+ gene product interacts with the product of cdc2+, but there is presently no evidence that Sucl+ plays a direct role in the cell division cycle. In the present study, we determined the nucleotide sequence of Sucl+, identified its protein coding sequence, ...

Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefmciafus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia culi of the toxin proteins and their NH2-and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by af6nity chromatography followed by cleavage from the GST carrier with thrombin. The LCs0 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.

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