Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Structure and expression of the human p68 RNA helicase gene.

Nuclear DEAD box protein p68 is immunologically related to SV40 large tumour antigen and both proteins possess RNA helicase activity. In this report, we describe the structural organisation of the human p68 gene and aspects of the regulation of its expression. Northern blot and primer extension analyses indicate that, although its level is variable, the p68 RNA helicase appears to be expressed from a single transcription start site in all tissues tested. Sequence analysis revealed that the p68 promoter harbours a 'TATA', a 'CAAT' and an initiator element and contains high affinity binding sites for Sp1, AP-2, CRE and Myc. This and functional promoter analyses in transient expression assays suggest that transcriptional regulation of the p68 gene is complex. Furthermore, there are indications that p68 expression is also regulated post-transcriptionally. Steady-state pools of poly(A)(+)RNA from human cells contain completely spliced p68 mRNA and alternatively spliced forms that contain introns 8-11 or 8-12 (from a total of 12 introns) and are not translated. Analysis of a conditionally p68-overproducing HeLa cell line points to negative autoregulation at the level of splicing, which is confirmed by a recently reported association of p68 with spliceosomes in human cells.

Membrane topology of CadA homologous P-type ATPase of Helicobacter pylori as determined by expression of phoA fusions in Escherichia coli and the positive inside rule.

The only experimental data available on the membrane topology of transition metal ATPases are from in vitro studies on two distinct P-type ATPases (CadA and CopA) of a gastric bacterium, Helicobacter pylori, both postulated to contain eight transmembrane domains (H1 to H8). In this study, H. pylori CadA ATPase was subjected to analysis of membrane topology in vivo by expression of ATPase-alkaline phosphatase (AP) hybrid proteins in Escherichia coli using a novel vector, pBADphoA. This vector contains an inducible arabinose promoter and unique restriction sites for fusion of DNA fragments to phoA. The phoA gene lacking sequences encoding its N-terminal signal peptide was linked to the C-terminal regions of the postulated five cytoplasmic and four periplasmic segments of the H. pylori pump. The results obtained by heterologous expression of ATPase-AP hybrid proteins showed consistence with a model of eight transmembrane domains. They also demonstrated that the H. pylori ATPase sequences are well assembled in the cytoplasmic membrane of E. coli, a neutralophilic bacterium. Cloning and amino acid sequence analysis of the homologous ATPase of Helicobacter felis further verified the topological model for the H. pylori pump analyzed here, although the degree of amino acid sequence identity varied between the corresponding transmembrane segments, from 25% for H1 up to 100% for H6. It was found that the topology of ATPase follows the 'positive inside rule'. With respect to the bioenergetic capacities of H. pylori, we discuss here the membrane potential as a possible factor directing insertion of ATPases in the cytoplasmic membrane of gastric bacteria.

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

Properties and function of the P type ion pumps cloned from Helicobacter pylori.

Three distinct P type pumps were cloned from H. pylori 69A. Two of these pumps, ATPase 439 and ATPase 948 (CopA), were isolated by gene library screening using DNA oligonucleotide primers. Amino acid similarities found for the predicted proteins were about 50% to Cd2+/Cu2+ pumps. Gene disruption mutagenesis rendered the H. pylori knockout mutants more sensitive to Zn2+ and Cd2+ (ATPase 439) or Cu2+ (CopA). Some of the ATPase 439-deficient mutants were negative for urease activity while the majority of the mutants remained positive. Functional diversity of the pumps was also reflected by the ion affinities found for N-terminal peptides of CopA to Cu2+ and of ATPase 439 to Ni2+, Cu2+ and CO2+. The membrane domain of the two pumps were experimentally shown to consist of eight membrane spans. When ATPase 439 was expressed under control of a tac promoter in Escherichia coli, vanadate-sensitive phosphate accumulation was observed cytochemically along the membrane of the host cells. The third P type pump (ATPase 115) which also exhibited homology to transition metal ATPase was identified by sequencing a library of H. pylori membrane genes. The hydropathy plot of this pump was very similar to the former H. pylori ATPases whereas the N-terminal ion binding region was distinct. It was concluded that, in H. pylori, the presence of three transition metal ATPases with distinct ion specificity contributes to the adaptive mechanisms for gastric survival.

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis.

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446-457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.

Cloning and membrane topology of a P type ATPase from Helicobacter pylori.

Southern blot screening of a genomic Helicobacter pylori library was employed to find a P type ATPase using a mixture of 16 DNA oligonucleotides coding for the DKTGT(I/L)T consensus sequence specific for the phosphorylation site of this family of ATPases. A positive clone, pRH439, was isolated and sequenced. The inserted 3.4-kb H. pylori DNA contained an intact open reading frame encoding a protein of 686 amino acids carrying the consensus sites for phosphorylation and ATP binding. The amino acid sequence exhibits a 25-30% identity with bacterial Cd2+ and Cu2+ ATPases. Genomic Southern blot analysis showed that this ATPase was present in all H. pylori strains examined, whereas it was not detectable in Campylobacter jejuni and other bacteria. The membrane topology of this ATPase was investigated using in vitro transcription/translation of fusion vectors to find signal anchor and/or stop transfer sequences. Eight regions of the H. pylori ATPase acted as signal anchor and/or stop transfer sequences and were ordered pairwise along the polypeptide chain placing the N and C-terminal amino acids in the cytoplasm. These transmembrane segments are contained between positions 73 and 92 (H1), 98 and 125 (H2), 128 and 148 (H3), 149 and 176 (H4), 309 and 327 (H5), 337 and 371 (H6), 637 and 658 (H7), and 659 and 685 (H8). The membrane domain of the ATPase, therefore, consists of at least four pairs of transmembrane segments with the phosphorylation site and ATP binding domain located in the large cytoplasmic loop between H6 and H7. The cytoplasmic domain contains several histidines and cysteines, perhaps indicative of divalent cation binding sites. There are several charged amino acids (3 Lys, 2 Glu, 2 Asp), predicted to be in the membrane domain mainly in H2, H3, and H4 and a Cys-Pro-Cys putative metal ion site in H6. The extracytoplasmic domain also has several charged amino acids (5 Glu, 1 Asp, 1 Lys, 1 Arg). It is likely that this novel protein is a heavy metal cation transporting ATPase and belongs to a family of P type ATPases containing eight transmembrane segments.