A high-density physical map of Sinorhizobium meliloti 1021 chromosome derived from bacterial artificial chromosome library.

As part of the European Sinorhizobium meliloti (strain 1021) chromosome sequencing project, four genomic bacterial artificial chromosome (BAC) libraries have been constructed, one of which was mainly used for chromosome mapping. This library consists of 1,824 clones with an average insert size of 80 kilobases and represents approximately 20-fold total genome coverage [6.8 megabases (Mbs)]. PCR screening of 384 BAC clones with 447 chromosomal markers (PCR primer pairs), consisting of 73 markers representing 118 genes (40 individual genes and 78 genes clustered in 23 operons), two markers from the rrn operon (three loci), four markers from insertion sequences (approximately 16 loci) and 368 sequence-tagged sites allowed the identification of 252 chromosomal BAC clones and the construction of a high-density physical map of the whole 3.7-Mb chromosome of S. meliloti. An average of 5.5 overlapping and colinear BAC clones per marker, correlated with a low rate of deleted or rearranged clones (0.8%) indicate a solid BAC contigation and a correct mapping. Systematic BLASTX analysis of sequence-tagged site marker sequences allowed prediction of a biological function for a number of putative ORFs. Results are available at. This map, whose resolution averages one marker every 9 kilobases, should provide a valuable tool for further sequencing, functional analysis, and positional cloning.

A 356-Kb sequence of the subtelomeric part of the MHC Class I region.

The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3. The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence. Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases. Most of the ESTs are clustered in two regions. In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes.

A low molecular weight DNA polymerase from wheat embryos.

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase beta. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.