We report the cloning of a novel murine cDNA, LEK1, that is related to human CENP-F and mitosin and more distantly to chicken CMF1. The proteins from these three organisms have significant homology, yet differ in their temporal, spatial, and subcellular localizations. The human proteins bind the kinetochore in mitotic cells, whereas the chicken protein is found only in skeletal and cardiac muscle and is developmentally regulated. Mouse LEK1 is a single copy gene that codes for two developmentally regulated transcripts. The LEK1 protein is expressed early and ubiquitously in mouse development and is generally down-regulated as development proceeds in a manner that correlates to a cessation of mitosis. In adult tissues, the LEK1 protein is detected exclusively in the pronucleus of the oocyte and was not observed in other actively dividing tissues. Subcellular localization revealed that the LEK1 protein in mitotic cells does not bind the kinetochore. From these data, we hypothesize that chicken CMF1, human CENP-F, mitosin, and mouse LEK1 are members of an emerging family of genes that have important and functionally distinct roles in development and cell division.

The nucleotide sequences, genetic organization, and distribution of plasmids pEU30 (30,314 bp) and pEL60 (60,145 bp) from the plant pathogen Erwinia amylovora are described. The newly characterized pEU30 and pEL60 plasmids inhabited strains isolated in the western United States and Lebanon, respectively. The gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 was most similar to IncL/M plasmids inhabiting enteric bacteria.Erwinia amylovora is the causal agent of fire blight, a serious disease of apple and pear that occurs in many locations throughout the world. The virulence plasmid pEA29 is conserved among all natural strains of E. amylovora, and several PCR assays for the identification of E. amylovora were developed on the basis of sequences within a 0.9-to 1

SUMMARY JC virus was previously isolated from the urine and from diseased brain of a patient with progressive multifocal leukoencephalopathy. The DNAs of these isolates, MAD-7 and MAD-8 respectively, were shown in this study to be mixtures of several different full-length genomes. By differences in their restriction endonuclease cleavage patterns, the genomes were categorized into two types. Type I DNA was defined as that typified by the prototype virus, MAD-l; Type II, that typified by viral DNA molecularly cloned from brain tissue of the MAD-7/8 patient (BrC-8 DNA). MAD-7 DNA was an approximately equal mixture of Type I and Type II; MAD-8 DNA appeared to be homogeneously Type II. These DNAs were molecularly cloned for further studies. Of 17 clones of recombinant MAD-7 (rMAD-7) DNA, nine were Type I and were identical to each other and to MAD-1 DNA. The other eight clones comprised five different species of Type II, none of which was identical to MAD-8 or to BrC-8 DNA. Among 17 recombinant MAD-8 (rMAD-8) clones there were two which contained Type I DNA, which had not been detected in the original viral DNA. The remaining rMAD-8 clones were of Type II. The differences among all these DNAs were primarily in the 'extra' PvulI sites and in the insertions and deletions in the hypervariable regulatory region of the genome (0-67 to 0.72 map unit). Possible explanations for multiple genomes in separate isolates from the same patient are discussed. It is concluded that the observed variation originated in vivo and not during isolation in vitro.

A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical isolates of Neisseria gonorrhoeae . The 70 S ribosomes from sensitive strains were sensitive to the effects of Str and Spc on synthesis directed by several synthetic polynucleotide messengers, whereas 70 S ribosomes from resistant strains were resistant to these same effects. In each case, the alteration was localized to the 30 S ribosomal subunit by studying antibiotic sensitivities of hybrid 70 S ribosomes formed by combining subunits from sensitive and resistant strains. No evidence was found for streptomycin- or spectinomycin-inactivating enzymes.

Nontypeable Haemophilus influenzae (NTHi) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our previous work has shown that NTHi biofilm maturation is coordinated by the production and uptake of autoinducer 2 (AI-2) quorum signals. To directly test roles for AI-2 in maturation and maintenance of NTHi biofilms, we generated an NTHi 86-028NP mutant in which luxS transcription was under the control of the xylA promoter (NTHi 86-028NP luxS xylA::luxS), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. The removal of xylose resulted in the interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript levels by real-time reverse transcription-PCR (RT-PCR) showed that luxS expression peaked as biofilms matured and waned before dispersal. Transcript profiling revealed significant changes following the induction of luxS, including increased transcript levels for a predicted family 8 glycosyltransferase (NTHI1750; designated gstA); this result was confirmed by real-time RT-PCR. An isogenic NTHi 86-028NP gstA mutant had a biofilm defect, including decreased levels of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections, we observed a significant decrease in the number of bacteria in the biofilm population (but not in effusions) for NTHi 86-028NP gstA compared to the parental strain. Therefore, we conclude that AI-2 promotes NTHi biofilm maturation and the maintenance of biofilm integrity, due at least in part to the expression of a probable glycosyltransferase that is potentially involved in the synthesis of the biofilm matrix.