Tandemly repeated satellite DNA ofDolichopoda schiavazzii: A test for models on the evolution of highly repetitive DNA

The evolutionary relationships among the Carnivora were studied in a phylogenetic analysis based on the complete mitochondrial cytochromeb gene. The study, which addressed primarily the relationships among the Caniformia, included 4 feliform and 26 caniform species, with 9 pinnipeds. The analysis identified five caniform clades: Canidae, Ailuridae (with the monotypic lesser panda), Musteloidea (Mustelidae+Procyonidae), Ursidae (including the giant panda), and Pinnipedia. The closest relatives of the Pinnipedia among terrestrial caniforms were not identified conclusively. Our analysis shows that the skunks are only distantly related to remaining mustelids (Mustelidae sensu stricto) and that the family Mustelidae, including the skunks, is paraphyletic. The relationship among the five caniform clades was unresolved, suggesting an evolutionary separation within a relatively short period of time. Based on distance values, we propose that this primary diversification took place 45 million years ago.     

Molecular and biochemical characterization of tomato farnesyl-protein transferase.

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.     

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.     

Assignment of the dystonia-parkinsonism syndrome locus, DYT3, to a small region within a 1.8-Mb YAC contig of Xq13.1.

A YAC contig was constructed of Xq13.1 in order to sublocalize the X-linked dystonia-parkinsonism (XDP) syndrome locus, DYT3. The contig spans a region of approximately 1.8 Mb and includes loci DXS453/DXS348/IL2R gamma/GJB1/CCG1/DXS559. For the construction of the contig, nine sequence-tagged sites and four short tandem repeat polymorphisms (STRPs) were isolated. The STRPs, designated as 4704#6 (DXS7113), 4704#7 (DXS7114), 67601 (DXS7117), and B4Pst (DXS7119) were assigned to a region flanked by DXS348 proximally and by DXS559 distally. Their order was DXS348/4704 #6/4704 #7/67601/B4Pst/DXS559. They were applied to the analysis of allelic association and of haplotypes in 47 not-obviously-related XDP patients and in 105 Filipino male controls. The same haplotype was found at loci 67601 (DXS7117) and B4Pst (DXS7119) in 42 of 47 patients. This percentage of common haplotypes decreased at the adjacent loci. The findings, together with the previous demonstration of DXS559 being the distal flanking marker of DYT3, assign the disease locus to a small region in Xq13.1 defined by loci 67601 (DXS7117) and B4Pst (DXS7119). The location of DYT3 was born out by the application of a newly developed likelihood method for the analysis of linkage disequilibrium.     

Backbone assignment,secondary structure and Protein A binding of an isolated,human antibody VH domain

Summary Antibody heavy chain variable domains (VH) lacking their light chain domain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with ¹⁵N or doubly labelled with both ¹⁵N and ¹³C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the ¹H and ¹⁵N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the ¹⁵N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the ¹³C-/¹⁵N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent -sheets formed by altogether nine antiparallel -strands. ³J_H ^NHC coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two -sheets of the VH. This -sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VH_III gene subgroup.Abbreviations CDR complementarity determining region - CHAPS [(cholamidopropyl)-dimethylammonio]-1-propanesulfonate - DQF-COSY double-quantum-filtered correlated spectroscopy - Fab antigen binding antibody fragment - Fc crystallisable antibody fragment - Fv heterodimer of VH and VL - H1 (2, 3) hypervariable loop 1 (2, 3) - IgG immunoglobulin G - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum correlation spectroscopy - HSQC heteronuclear single quantum correlation spectroscopy - scFv single chain Fv - TOCSY total correlation spectroscopy - TPPI time-proportional phase incrementation - VH antibody heavy chain variable region - VL antibody light chain variable region. Mutants are denoted by the wild-type amino acid (one-letter code), follwed by the residue number and the new amino acid     

Nonrandom chromosome variation and morphogenic potential in cell lines of bread wheat (Triticum aestivum L.).

A cytogenetical analysis of 18 cell lines, 9 microspore derived, 6 anther derived, and 3 immature-embryo derived, of bread wheat (Triticum aestivum L.) varying in their morphogenic potential was undertaken. Chromosome variation, both structural and numerical, was detected in all lines studied. Variation was present and, in some cases quite extensive, in the earliest samples taken (only 12 weeks after initiation of the suspensions). Within any culture, the pattern and extent of variation changed throughout the course of the study and cells with a euploid constitution generally decreased in frequency with culture age. Among the nine microspore-derived suspensions, morphogenic lines generally showed a more restricted range of chromosome numbers and higher proportions of euploid cells than nonmorphogenic lines. The patterns of distribution of chromosome numbers among the anther-derived cultures were similar to those of the microspore-derived lines but the correspondence between instability and regenerative capacity was less. The immature embryo derived lines, which were neither regenerable nor morphogenic, were all unstable. The anther-derived lines were sampled over several months to determine whether loss of morphogenic potential was related to changes in chromosome instability of specific lines. Analysis of the "elite" line Fl.7, initially capable of regenerating green plants, showed that substantial decreases in the frequencies of normal euploid cells (from 45 to 5%) occurred over the period when morphogenic capacity was lost. However, whether the chromosome instability resulted in loss of morphogenicity or vice versa was not clarified. C-banding analyses of lines Fl.7 and C82d indicated that instability was not random with respect to the three genomes (A, B, and D) of wheat nor to the different chromosomes within the genomes. Chromosomes of the B genome were most often lost or involved in rearrangements, with breakpoints located at, or near, the heterochromatic blocks. Because of the heterogeneity of the cell lines, extensive analyses of large numbers of cells would be required before it would be possible to determine whether loss of morphogenic potential arises as a result of specific chromosome loss(es).     

Congenic strains reveal effects of the epilepsy quantitative trait locus,El2, separate from other El loci

Congenic mouse strains made by transferring epilepsy predisposing alleles El1, El2, and El3 from the EL/Suz strain to the ABP/Le recipient were tested for seizure frequency following gentle rhythmic stimulation. Mice homozygous for El2, but not El1 or El3, experienced seizures much more frequently than ABP controls, while respective El1 homozygotes and El2 heterozygotes had only a modest increase over ABP, and El3 homozygotes showed no increase. Association between marker genotypes and seizure trequency in small intra-strain crosses showed that the phenotypic effects of El2 map to the selected interval, and that segregation of El2 accounts for virtually all genetic effects. However, in separating El2 from other EL susceptibility alleles, the seizure frequency phenotype was weaker and less heritable than in crosses between parental strains. These results confirm El2 as an important QTL and show that it has significant phenotypic effects in the absence of other EL-derived alleles, including El1. In addition, the present localization of El2 on Chr 2 suggests several potential candidate genes for El2, including the subunit of phospholipase-C. The approach to dissecting complex traits by making congenic strains for individual QTL is discussed.Center for Medical Genetics, Marshfield Medical Research Foundation, Marshfield, Wisconsin, 54449, USA     

Genomic organization and sequence of the rat major histocompatibility complex class Ia gene RT1.A u

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X82669     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ^?) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.