Unique Telomeric Expression Site of Major-Surface-Glycoprotein Genes of Pneumocystis carinii

Major cell surface glycoproteins (MSG) of Pneumocystis cariniiplay a crucial role in the host-parasite interactions involvedin P. carinii pneumonia in AIDS patients. Genes encoding MSGsare repeated, highly polymorphic, and distributed among allof the 14-15 chromosomes. Here we show, by BAL-31 exonucleasecleavage and DNA cloning experiments, that the unique expressionsite (previously termed UCS) of MSG genes located in the 500-kbchromosome is telomeric. The 11-kb genomic UCS fragment isolatedand sequenced in this study contained one MSG coding sequence(termed msg105), subtelomeric repetitive sequences and telomere-specifictandem repeats of TTAGGG oriented 5' to 3' towards the DNA end.Despite the N-terminal polymorphism, the C-terminal one-thirdsequence of MSG105 was identical to one of the known MSG-cDNAs,suggesting homologous recombination within the MSG coding sequences.These features closely resemble the Variant Surface Glycoproteinsystem of the protozoan parasite Trypanosoma brucei, suggestingthat the genetic heterogeneity of MSGs is generated by recombinationbetween the UCS expression site and multiple MSG genes by meansof reciprocal exchange or gene conversion.     

Multi-Gene Family of Major Surface Glycoproteins of Pneumocystis carinii: Full-Size cDNA Cloning and Expression

The major surface glycoprotein (MSG) of Pneumocystis cariniiplays a crucial role in the fatal pneumonia caused by this organismin AIDS patients. A cDNA encoding a full-length MSG polypeptidewas isolated from a phage library of rat-derived P. cariniicDNAs. The deduced MSG, referred to as the MSG5 subtype, isa 120,765-Da protein composed of 1,076 amino acids and containsan anchoring hydrophobic sequence at the C-terminus of the protein.Sequence analyses of cloned MSG-cDNAs revealed an MSG-gene familywith 70% protein sequence identity between subtypes. P. cariniikaryotype hybridization analyses indicated that the MSG genefamily members are scattered throughout most of the P. cariniichromosomes. These recombinant MSG proteins reacted with theantiserum from P. carinii-infected rats, as expected, and antiserumgenerated against P. carinii-infected mice, indicating the existenceof common determinants in MSG polypeptides. The family of MSGproteins is rich in cysteine residues and these cysteine arehighly conserved in all MSG subtypes regardless of species specificity,suggesting the structural and/or functional importance of thesecysteine. The pathobiological significance of the MSG gene familyand its sequence diversity in P. carinii is discussed.     

Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24

A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5α using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.     

Cloning, Characterization, and Possible Origin of the Prevotella loescheii dnaK Homolog

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella. Received: 8 June 2000 / Accepted: 11 August 2000     

Construction of a Contiguous 874-kb Sequence of the Escherichia coli-K12 Genome Corresponding to 50.0-68.8 min on the Linkage Map and Analysis of Its Sequence Features

The contiguous 874.423 base pair sequence corresponding to the50.0–68.8 min region on the genetic map of the Escherichiacoli K-12 (W3110) was constructed by the determination of DNAsequences in the 50.0–57.9 min region (360 kb) and twolarge (100 kb in all) and five short gaps in the 57.9–68.8min region whose sequences had been registered in the DNA databases.We analyzed its sequence features and found that this regioncontained at least 894 potential open reading frames (ORFs),of which 346 (38.7%) were previously reported, 158 (17.7%) werehomologous to other known genes, 232 (26.0%) were identicalor similar to hypothetical genes registered in databases, andthe remaining 158 (17.7%) showed no significant similarity toany other genes. A homology search of the ORFs also identifiedseveral new gene clusters. Those include two clusters of fimbrialgenes, a gene cluster of three genes encoding homologues ofthe human long chain fatty acid degradation enzyme complex inthe mitochondrial membrane, a cluster of at least nine genesinvolved in the utilization of ethanolamine, a cluster of thesecondary set of 11 hyc genes participating in the formate hydrogenlyasereaction and a cluster of five genes coding for the homologuesof degradation enzymes for aromatic hydrocarbons in Pseudomonasputida. We also noted a variety of novel genes, including twoORFs, which were homologous to the putative genes encoding xanthinedehydrogenase in the fly and a protein responsible for axonalguidance and outgrowth of the rat, mouse and nematode. An isoleucinetRNA gene, designated ileY , was also newly identified at 60.0min.     

Molecular Genetic Distinction of Pneumocystis carinii from Rats and Humans

Pneumocystis carinii from rats and from humans were compared with respect to electrophoretic karyotype, presence of DNA sequences known to be repeated in rat-derived P. carinii, overall DNA sequence homology, and the sequences at two genetic loci. The organisms from each host species were different in each respect. Neither of two repeated DNAs from rat-derived P. carinii was found in the genome of human-derived organisms, and total DNA from rat-derived P. carinii failed to hybridize to human-derived P. carinii DNA. The sequences of the α-tubulin genes from the two P. carinii were strikingly different and the base composition of the α-tubulin gene from rat-derived P. carinii was rich in adenine and thymine, while the base composition of this gene from human-derived P. carinii was rich in guanine and cytosine. The sequence from the 18S rRNA gene of human-derived P. carinii was twice as divergent from that of rat-derived P. carinii as the sequence from the corresponding region of Candida albicans was from that of Candida tropicalis. These data show that rats and humans can harbor distinct types of P. carinii that are sufficiently different to suggest that P. carinii from the two hosts could be different species.     

Cloning and Sequencing of a 36-kb Region of the Bacillus subtilis Genome between the gnt and iol Operons

Within the framework of an international project for the sequencingof the entire Bacillus subtilis genome, a 36-kb chromosome segment,which covers the region between the gnt and iol operons, hasbeen cloned and sequenced. This region (36447 bp) contains 33complete open reading frames (ORFs; genes) including the fourgnt genes and one partial gene. A homology search for the productsof the 33 complete ORFs revealed significant homology to knownproteins in 16 of them such as tetracycline resistance protein(Clostridium perfringens), asparagine synthetase (Arabidopsisthaliana), aldehyde dehydrogenase (Pseudomonas oleovorans),2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (P. paucimobilis),heat shock protein HtpG (Escherichia coli), galactose-protonsymporter (E. coli), auxin-induced protein (common tobacco),glucitol operon repressor (E. coli) and methylmalonate-semialdehydedehydrogenase (P. aeruginosa). Unlike the regions we sequencedso far, this region contained two short sequence multiplications:one was a tandem sequence duplication (409 and 410 bp), andthe other a triplication consisting of two highly conserved118-bp tandem sequences preceded by a less conserved similarsequence (129 bp). The reasons for the presence of these sequencemultiplications in the gnt to iol region were deduced.     

A Tandem Repeat of Rat-derived Pneumocystis carinii Genes Encoding the Major Surface Glycoprotein

ABSTRACT. A fragment from the genome of rat-derived Pneumocystis carinii was found to contain two MSG genes arranged as a direct repeat. The sequences from one gene (MSG B), the region between the two genes, and part of the second gene (MSG A) were determined. The two MSG genes were not identical in sequence. The open reading frames of MSG A and MSG B encode non-identical proteins, both of which are similar to that encoded by a previously published cDNA. The MSG B gene sequence showed no evidence of introns. The 5'and 3'untranslated regions of the MSG gene pair were highly conserved, but the regions immediately upstream of the open reading frames of MSG A and B were different from the region upstream of a previously characterized MSG cDNA. Primers designed to extend upstream of the 5'end of MSG and downstream of the 3'end of MSG were used in a polymerase chain reaction with total genomic P. carinii DNA as template. Presumptive intergenic amplification products from this reaction were cloned and sequenced. The sequences of these regions were similar but distinct, indicating that tandem arrangement of MSG genes is a common organizational motif.     

Nucleotide Sequence of cDNA for Concanavalin A from Canavalia gladiata Seeds

We have determined the nucleotide sequence of Con A cDNA forconcanavalin A (Con A) from Canavalia gladiata using a plasmid(pCONAl) that was isolated previously [Eur. J. Bio-chem. (1988)170: 515-520]. This sequence contains a 870-bp open readingframe, a 63-bp 5'-un-translated region and a 99-bp 3'-untranslatedregion. DNA blot analysis suggested that Con A is encoded bya small gene family. In contrast to the case of canavalin, thenucleotide sequence and the genomic organization of Con A geneare highly conserved between C. gladiata and C. ensifor-mis. (Received August 4, 1988; Accepted November 21, 1988)     

Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae

The Saccharomyces cerevisiae MNN4 gene, which is involved inmannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide,has been cloned from a lambda phage containing a yeast chromosomeXI DNA fragment The MNN4 ORF encodes a protein of 1178 aminoacids. The deduced amino acid sequence shows a topology of typeII membrane proteins and has a unique repeated sequence of lysineand glutamic acid at the C-terminus. Disruption and overexpressionof MNN4 led to a decrease and increase, respectively, of themannosylphosphate content in cell wall mannans prepared fromboth mnn4 and wild type strains. A dramatic decrease of mannosylphosphateoccurs in     

Sequence Analysis of a 685-kb Genomic Region on Chromosome 3p22-p21.3 That Is Homozygously Deleted in a Lung Carcinoma Cell Line

Frequent chromosomal aberrations and/or losses of heterozygosityinvolving the short arm of chromosome 3 in carcinomas of thelung, kidney and other tissues imply that multiple putativetumor suppressor genes may be present on this chromosomal arm.To search for one of these genes, we determined DNA sequencesin the genomic region at 3p22–21.3 where we had previouslydetected a homozygous deletion in a lung cancer cell line. TheDNA sequence results of an about 685-kb region indicated thatthe size of the homozygously deleted segment was 638,489 bp,in which we identified only four genes including the integrinRLC and the trans-Golgi p230 genes, both reported previously.The predicted amino acid sequences of one of the two novel genesshowed high homology to villin, a human cytoskeleton protein;those of the other gene, termed HYA22, revealed significanthomology to YA22, a hypothetical protein predicted from DNAsequences of Schizosaccharomyces pombe. The computer programsHEXON or GRAIL were able to predict three-fourths of the exons;the smallest exon predicted by either program was 46 base pairs.Repetitive sequences contained in the genomic region included151 copies of the Alu sequence (1 copy/every 4.5 kb), 19 copiesof the L1 sequence (1 copy/every 36 kb) , and 10 copies of theTHE sequence.     

A herpesvirus saimiri protein required for interleukin-2 independence is associated with membranes of transformed T cells.

A region of the herpesvirus saimiri genome encoding an mRNA with two open reading frames (ORFs) has been identified to be essential for transformation of T cells. Deletion of either ORF resulted in the loss of transforming ability. ORF-1 has been shown to code for a collagen-like oncoprotein. This study shows for the first time that the bicistronic mRNA can translate a 32-kDa protein from ORF-2. Polyclonal serum to ORF-2 was generated by using a glutathione fusion protein. Using this antiserum, ORF-2 was localized in cell membranes and is expressed on the outer cell membrane. The half-life of this membrane protein was found to be about 5.5 h. Limited sequence similarity was found between ORF-2 and interleukin-11; however, no secretion of ORF-2 protein was detected in supernatants from transformed cells. Further studies are required to investigate the potential interaction with the interleukin-11 receptor.     

Model Organisms: New Insights: Into Ion Channel and Transporter Function. Caenorhabditis elegans ClC-type chloride channels: novel variants and functional expression

Six ClC-type chloridechannel genes have been identified in Caenorhabditiselegans, termed clh-1 through clh-6. cDNAsequences from these genes suggest that clh-2,clh-3, and clh-4 may code for multiple channelvariants, bringing the total to at least nine channel types in thisnematode. Promoter-driven green fluorescent protein (GFP) expression intransgenic animals indicates that the protein CLH-5 isexpressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells,and the remaining isoforms vary from those restricted to a single cellto those expressed in over a dozen cells of the nematode. In an Sf9cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did notform functional plasma membrane channels. In contrast, both CLH-1 andCLH-3b produced strong, inward-rectifying chloride currents similar tothose arising from mammalian ClC2, but which operate over differentvoltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene familyprovides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.

     

Sequence Organization of Simple, Highly Repetitive DNA Elements in Brassica species

The amphidiploid (AACC) nuclear genome of Brassica napus (oil-seedrape) contains c. 5 ? 10⁵ copies of a simple, highly repetitiveDNA element; each repeat is 176 or 177 base pairs long and isdefined by Hind III cutting sites. The diploid (AA) Brassicacampestris (turnip) possesses a very similar repetitive DNA,the consensus sequence of which does not differ from that inB. napus. The 176/177 bp unit consists of three 59 bp sub-units,defined by vestigial EcoRII sites. Analysis of the distributionof variants from consensus in adjacent and non-adjacent unitsprovides evidence for homogenization of sequences by the fixationof independent mutations and for tandem duplication of units.Within units, there is also evidence for inversion and tandemduplication of short (5–8 bp) motifs. Previously published data show that 176/177 base pair repetitiveDNA elements, defined by Hind III cutting sites, are also presentin Sinapis and Raphanus. There is a sequence homology betweenBrassica and Sinapis, and between Brassica and Raphanus, of75%. Sequence homology between Raphanus and Sinapis is 73%. Key words: Repetitive DNA, Brassica, Cruciferae     

Characterization of Pneumocystis carinii PHR1, a pH-Regulated Gene Important for Cell Wall Integrity

Pneumocystis carinii remains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region coding GSC-1, a gene mediating β-glucan cell wall synthesis in P. carinii. The predicted amino acid sequence of this new gene, termed P. carinii PHR1, exhibited 38% homology to Saccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology to Candida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression of P. carinii PHR1 was examined. As in C. albicans, P. carinii PHR1 expression was repressed under acidic conditions but induced at neutral and more alkaline pH. PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties with P. carinii culture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression of P. carinii genes in related fungi to address functional correlates of P. carinii-encoded proteins. Therefore, the potential role of P. carinii PHR1 in cell wall integrity was examined by assessing its ability to rescue an S. cerevisiae gas1 mutant with absent endogenous Phr1p-like activity. Interestingly, P. carinii PHR1 DNA successfully restored proliferation of S. cerevisiae gas1 mutants under lethal conditions of cell wall stress. These results indicate that P. carinii PHR1 encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.