Complete Genome Sequence of Anaeromyxobacter sp. Fw109-5, an Anaerobic, Metal-Reducing Bacterium Isolated from a Contaminated Subsurface Environment.

We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacterium's genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation.

Dictyostelium discoideum contains a single-copy gene encoding a unique subtype of histone H1.

A Dictyostelium discoideum genomic library was screened using a degenerate oligodeoxyribonucleotide derived from the peptide, GPKAPT, obtained from the N terminus of purified histone H1. Two identical H1 clones were isolated. Comparative sequence data reveal a typical H1 three-domain structure with considerable homology to the globular domain of higher eukaryotic H1 histones, especially to plant H1 histones. Southern blot analysis shows that this gene is probably a single-copy gene, and suggests that any other H1 gene(s), if present, must be very different in sequence. Amino acid (aa) sequence comparison of the globular core of D. discoideum H1 to the consensus globular core reveals the absence of a 6-aa motif, GXGXXG, from D. discoideum. This motif matches the consensus for a putative nucleotide-binding loop, which is also absent in plant H1 histones like Arabidopsis thaliana, pea and wheat.

Structure of the macronuclear polyubiquitin gene in Euplotes.

The hypotrichous ciliate, Euplotes eurystomus, contains both a transcriptionally inactive micronucleus (MIC) and a transcriptionally active macronucleus (MAC) in the same cell. MAC DNA is small (0.5-20 kb), linear and highly amplified. Each DNA fragment consists of two telomeres, a single coding region, and the necessary control elements to regulate gene transcription and replication. The polyubiquitin gene consists of 898 bp, plus 28 bp of double-stranded and 14 bases of single-stranded DNA of the telomeric repeat G4T4 at each end. The coding region exists as three copies of the ubiquitin gene (690 bp) fused in a head-to-tail arrangement as in other organisms. The stop codon is TAA, as in other Euplotes genes, and is not the rare glutamine codon used in most other ciliates. The 3' nontranslated region contains two presumptive poly(A) addition sites; the 5' nontranslated region possesses two putative TATA boxes, several imperfect direct and inverted repeats, and a possible origin of replication. Nucleosome positioning studies reveal four tightly packed nucleosomes and a non-nucleosomal area containing the probable 5' control region as well as part of the coding region. The 5' area does not contain any DNAse I hypersensitive sites. Although the telomeres are protected from exonuclease digestion, they are not as well protected as Oxytricha telomeres against endonucleases and cleavage by methidium propyl Fe2+ EDTA.

The 5S RNA gene minichromosome of Euplotes.

The macronucleus of the ciliated protozoan Euplotes eurystomus contains about 10(6) copies of a single type of 5S ribosomal RNA gene. This 5S gene DNA is only 930 bp long, is flanked by telomeres, and contains a single coding region of 120 bp which serves as a template for transcription in vivo and in vitro. The 5S gene minichromatin possesses four positioned nucleosomes and hypersensitive cleavage sites in the telomeric regions.

Predicting protein folding classes without overly relying on homology.

An important open problem in molecular biology is how to use computational methods to understand the structure and function of proteins given only their primary sequences. We describe and evaluate an original machine-learning approach to classifying protein sequences according to their structural folding class. Our work is novel in several respects: we use a set of protein classes that previously have not been used for classifying primary sequences, and we use a unique set of attributes to represent protein sequences to the learners. We evaluate our approach by measuring its ability to correctly classify proteins that were not in its training set. We compare our input representation to a commonly used input representation--amino acid composition--and show that our approach more accurately classifies proteins that have very limited homology to the sequences on which the systems are trained.

Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining.

Four histone H1 subtypes and H1(0) were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzyme-linked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and alpha-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluorescence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.

Point mutations in the murine fumarylacetoacetate hydrolase gene: Animal models for the human genetic disorder hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah(6287SB) allele is a missense mutation in exon 6, and Fah(5961SB) is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah(6287SB) and Fah(5961SB) mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah(5961SB) and Fah(6287SB) as mouse models for acute and chronic forms of human HT1, respectively.

Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis.

Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.