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.     

Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.     

Vitellogenin motifs conserved in nematodes and vertebrates

Summary Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.     

Pneumocystis carinii and Pneumocystis wakefieldiae in Wild Rattus norvegicus Trapped in Thailand

ABSTRACT. This work reports for the first time the presence of two Pneumocystis species in wild Rattus norvegicus specimens from Thailand. Pneumocystis DNA was detected in 57.7% (15/26) wild rats without apparent association with typical pneumocystosis. Pneumocystis carinii was found alone in five rats (19.2%), Pneumocystis wakefieldiae was detected alone in six rats (23.1%), and two rats were infected by both species (7.7%). In addition, a new P. wakefieldiae variant sequence has been identified in three wild R. norvegicus specimens caught in the same geographical area. The high frequency of Pneumocystis in wild rats documented in this study and the apparent scarcity of severe pneumocystosis were consistent with an efficient circulation of rat Pneumocystis species in ecosystems.     

Molecular sequencing and analysis of soluble methane monooxygenase gene clusters from methanotroph <Emphasis Type="Italic">Methylomonas</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> GYJ3

Summary A methanotroph Methylomonas sp. GYJ3 was isolated, whose sMMO genes and 16S rDNA were sequenced and analysed, demonstrating that the bacterium might be a type I methanotroph (γ-Proteobacteria) and was closer to Methylomonas sp. KSWIII/KSPIII. This result was consistent with the result previously determined by biochemistry and morphological taxonomy. Sequence comparison among six open reading frames and the deduced amino acid sequences of the sMMO genes from six strains revealed that the strain GYJ3 had highly conserved regions in MMOX with other strains, amounting to 78–99% homology at protein level and 71–97% homology at DNA level. Highly conserved sequences lay in two iron-binding regions. Furthermore, scanning electron microscopy of the strain GYJ3 showed rod shapes with a slightly bent configuration on the even surfaces and with plump bodies.     

Nucleotide sequence of psbC,the gene encoding the CP-43 chlorophyll a-binding protein of Photosystem II,in the cyanobacterium Synechocystis 6803

The nucleotide sequence for the Photosystem II gene psbC has been determined for the cyanobacterium Synechocystis 6803. The gene overlaps the last 50 bases of the psbD gene, and both genes are transcribed in the same direction, but read in different frames. This arrangement is identical to that found in all chloroplast genomes for which psbC has been sequenced. The Synechocystis nucleotide sequence is 70% homologous to the tobacco gene and the predicted amino acid sequence shows 85% homology. A possible alternative translation start site for psbC has been conserved between seven plant sequences and the cyanobacterial sequence. The hydropathy plot for the cyanobacterial protein is very similar to plots determined for six plant species. Pairs of histidines that may play a role in binding chlorophyll are conserved between the cyanobacterial and plant amino acid sequences.     

T-protein of the glycine decarboxylase multienzyme complex: evidence for partial similarity to formyltetrahydrofolate synthetase

We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum. The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins. Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes. This suggests that the T-protein sequence is not as unique as previously thought.     

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.     

Evolutionary History of 4.5SI RNA and Indication That It Is Functional

To date, the small nuclear 4.5S_I RNA has only been studied in the rat (Rattus norvegicus). Combining PCR and hybridization analyses, we have revealed 4.5S_I RNA homologues sequences in the genomes of four myomorph rodent families (Muridae, Cricetidae, Spalicidae, and Rhizomyidae), and not in other myomorph families (Dipodidae, Zapodidae, Geomyidae, and Heteromyidae) or sciuromorph and caviomorph rodents. By Northern-hybridization, 4.5S_I RNA has been detected in the common rat (R. norvegicus, Muridae), golden hamster (Mesocricetus auratus, Cricetidae), and Russian mole rat (Spalax microphthalmus, Spalacidae), but not in the related great jerboa (Allactaga jaculus, Dipodidae) or in four non-myomorph rodent species tested. cDNA derived from 4.5S_I RNA of M. auratus and S. microphthalmus has been cloned and sequenced. The hamster RNA is found to differ from rat 4.5S_I RNA by only one nucleotide substitution. For the mole rat, two variants of 4.5S_I RNA are detected: short (S) and long (L) with length 101 and 108 nt, respectively. The L variant differs from the S variant as well as from murid and cricetid 4.5S_I RNAs by both a 7 nt insertion and a varying number of nucleotide substitutions. The sequence similarity between the spalacid S-variant and murid/crecitid variants of 4.5S_I RNA is 90%. Judging from species distribution, 4.5S_I RNA genes emerged during the same period of time as the related short interspersed element B2 arose. This occurred after the divergence of Dipodidae lineage but before the branching of Spalicidae/Rhizomyidae lineage from a common myomorph rodent stem. S variant genes seemed to emerge in a common ancestor of spalacids and rhizomyds whereas L variant genes formed in spalacids following the divergence of these two families. The low rate of evolutionary changes of 4.5S_I RNA, at least, in murids and cricetids (6 × 10⁻⁴ substitutions per site per million years), suggests that this RNA is under selection constraint and have a function. This is a remarkable fact if the recent origin and narrow species distribution range of 4.5S_I RNA genes is taken into account. Genes with narrow species distribution are proposed to be referred to as stenogenes. Received: 11 December 2000 / Accepted: 27 August 2001     

Protein adaptation to low temperatures: a comparative study of α-tubulin sequences in mesophilic and psychrophilic algae

The α-tubulin genes from two psychrophilic algae belonging to the genus Chloromonas (here named ANT1 and ANT3) have been isolated and sequenced. The genes ant1 and ant3 contain 4 and 2 introns, respectively. The coding DNA sequences are 90% identical but the degree of isology is very high at the polypeptide level (more than 97% strict identities). The ANT1 and ANT3 α-tubulin amino acid sequences were compared to the corresponding sequence of the mesophilic alga Chlamydomonas reinhardtii. Of the 15 substitutions detected in ANT1 and/or ANT3, 5 are common to both psychrophilic algae. The recorded substitutions have been analyzed in terms of cold adaptation on the basis of the available three-dimensional structure of the α,β-tubulin heterodimer from pig brain. Most of these are subtle changes, but two substitutions, M268V and A295V occurring in the region of interdimer contacts, could be of great significance for the cold stability of Antarctic algae microtubules due to the fact that the entropic control of microtubule assembly is particularly high in cold adaptes species. Received: December 24, 1998 / Accepted: April 2, 1999     

Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D63675 and D63676.     

Detection of a novel spotted fever group rickettsia in <Emphasis Type="Italic">Amblyomma parvum</Emphasis> ticks (Acari: Ixodidae) from Argentina

The present study evaluated the rickettsial infection in Amblyomma parvum ticks collected in Northwestern Córdoba Province, Argentina. Each tick was subjected to DNA extraction and tested by polymerase chain reaction (PCR) targeting fragments of the rickettsial genes gltA and ompB. Nine (69.2%) out of 13 adult ticks yielded expected PCR products for the two rickettsial genes. Products from the ompB PCR were sequenced, generating DNA sequences 100% identical for the nine PCR-positive ticks. Three of these ticks were tested in another battery of PCR targeting fragments of the rickettsial genes gltA, htrA, and ompA. Products from the gltA, htrA, and ompA PCRs were sequenced generating DNA sequences 100% identical for the three PCR-positive ticks. The rickettsia detected in the A. parvum ticks was designated as Rickettsia sp. strain Argentina. Phylogenetic analyses performed with partial sequences of the rickettsial genes gltA, htrA, ompB, and ompA showed that Rickettsia sp. strain Argentina belonged to the spotted fever group, being distinct from all known Rickettsia species and genotypes available in GenBank, representing possibly a new Rickettsia species. This was the first evidence of rickettsial infection in the tick A. parvum, and the third report of rickettsial infection among the Argentinean tick fauna. The role of Rickettsia sp. strain Argentina as a human pathogen is unknown. Further studies are needed to obtain tissue-cultured isolates of Rickettsia sp. strain Argentina, in order to better characterize it and to determine its taxonomic status as a new species.     

On the Origins of a Vibrio Species

Thirty-two genome sequences of various Vibrionaceae members are compared, with emphasis on what makes V. cholerae unique. As few as 1,000 gene families are conserved across all the Vibrionaceae genomes analysed; this fraction roughly doubles for gene families conserved within the species V. cholerae. Of these, approximately 200 gene families that cluster on various locations of the genome are not found in other sequenced Vibrionaceae; these are possibly unique to the V. cholerae species. By comparing gene family content of the analysed genomes, the relatedness to a particular species is identified for two unspeciated genomes. Conversely, two genomes presumably belonging to the same species have suspiciously dissimilar gene family content. We are able to identify a number of genes that are conserved in, and unique to, V. cholerae. Some of these genes may be crucial to the niche adaptation of this species.     

Antigenic properties of recombinant glycosylated and nonglycosylatedPneumocystis carinii glycoprotein A polypeptides expressed in baculovirus-infected insect cells

Since a continuous culture system is not yet available for the opportunistic fungal pathogenPneumocystis carinii, obtaining suitable amounts of purifiedP. carinii antigens free of mammalian-host lung contaminants is difficult. Hence, production of recombinant antigen possessing epitopes found in nativeP. carinii antigens is critical for immunological studies. We utilized the baculovirus expression vector system (BEVS) in insect cells to determine whether B-cell epitopes present in the protein core of a nativeP. carinii surface glycoprotein were conserved in the recombinant polypeptide, and to investigate its glycosylation by insect cells. B-cell epitopes were retained, but the insect cells appeared to hyperglycosylate the recombinant protein.