Internal transcribed spacer regions of rRNA genes of Pneumocystis carinii from monkeys.

Analysis of sequence variations among isolates of Pneumocystis carinii f. sp. macacae from 14 Indian rhesus monkeys (Macaca mulatta) at the internal transcribed spacer (ITS) regions of the nuclear rRNA gene was undertaken. Like those from P. carinii f. sp. hominis, the ITS sequences from various P. carinii f. sp. macacae isolates were not identical. Two major types of sequences were found. One type of sequence was shared by 13 isolates. These 13 sequences were homologous but not identical. Variations were found at 13 of the 180 positions in the ITS1 region and 28 of the 221 positions in the ITS2 region. These sequence variations were not random but exhibited definite patterns when the sequences were aligned. According to this sequence variation, ITS1 sequences were classified into three types and ITS2 sequences were classified into five types. The remaining specimen had ITS1 and ITS2 sequences substantially different from the others. Although some specimens had the same ITS1 or ITS2 sequence, all 14 samples exhibited a unique whole ITS sequence (ITS1 plus ITS2). The 5.8S rRNA gene sequences were also analyzed, and only two types of sequences that differ by only one base were found. Unlike P. carinii f. sp. hominis infections in humans, none of the monkey lung specimens examined in this study were found to be infected by more than one type of P. carinii f. sp. macacae. These results offer insights into the genetic differences between P. carinii organisms which infect distinct species.

Characterization of the gene encoding a histidine and aspartic acid-rich protein from Pneumocystis carinii.

A cDNA clone derived from Pneumocystis carinii contained an unusual sequence (GTGATG)2(ATGGTG)4(ATG)4 and many GAT repeats. It was found to encode a histidine and aspartic acid-rich protein (HARP). The complete cDNA contained an 888-bp open reading frame encoding a putative protein of 32.6 kDa. The deduced HARP protein contained 39 aspartic acid and 22 histidine residues. The genomic copy of the HARP gene (1203 bp in length) was found to contain 3 small introns of 46, 44, and 38 bp, respectively. HARP was predicted by computer programs to be a plasma membrane protein with nickel-binding activity.

Down-regulation of GATA-2 transcription during Pneumocystis carinii infection.

Differences in gene expression between Pneumocystis carinii-infected and noninfected rats were examined. Total RNA was isolated from homogenized rat lungs and then subjected to differential display with combinations of oligo(dT) and various arbitrary PCR primers. Approximately 50 differentially expressed bands were observed. Several of these DNA bands were isolated, reamplified, and cloned. The cloned DNA fragments were used as probes to perform Northern hybridization on RNA from P. carinii-infected and noninfected rat lungs. One clone was found to react with a 3-kb mRNA from noninfected but not from P. carinii-infected rat lung, suggesting that the gene represented by this clone was down-regulated during P. carinii infection. The nucleotide sequence of this clone was determined and found to be 97% homologous to the mouse GATA-2 transcription factor. In situ hybridization using RNA probes derived from this clone revealed that alveolar macrophages, resident lung monocytes, and bronchial epithelial cells express the GATA-2 gene in the lung.

Development and evaluation of a molecular viability assay for Pneumocystis carinii.

Despite recent declines in incidence, Pneumocystis carinii pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While P. carinii DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for P. carinii. This method is based upon the detection of P. carinii mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 10(6) trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing P. carinii from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable P. carinii collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.

Identification of domains of the human A1 adenosine receptor that are important for binding receptor subtype-selective ligands using chimeric A1/A2a adenosine receptors.

To provide new insights into the regions of the human A1 adenosine receptor (A1AR) involved in ligand binding, a series of chimeric human A1 and rat A2a adenosine receptors (A1/A2a) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the A2aAR agonist [3H]CGS-21680, the A1AR agonist [3H]2-chloro-N6-cyclopentyladenosine (CCPA), and the A1AR antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the A2aAR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the A1AR (A1TM1/A2a), [3H]CGS-21680 and [3H]CCPA binding was detectable. When an A1TM1-2/A2a construct was studied, [3H]CGS-21680 binding was lost and [3H] DPCPX binding appeared. Saturation studies using [3H]CCPA revealed that the A1TM1/A2a construct had low affinity. However, with the subsequent addition of A1AR TMs 2-4 receptor affinity improved markedly. Saturation studies using [3H]DPCPX also revealed that the TMs 1-4 of the A1AR conferred wild-type receptor affinity. When the ligand binding properties of A1TM1-4/A2a, A1TM1-6/A2a, and wild type A1AR constructs were directly compared, no differences were found using 10 different compounds. When truncated A1ARs that extended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data suggest that the important determinants for A1AR agonist and antagonist binding and ligand specificity are present in TMs 1-4.

The human A1 adenosine receptor: ligand binding properties, sites of somatic expression and chromosomal localization.

The A1 adenosine receptor (A1AR) exerts important biological effects in the mammalian biology. To provide insights into the role A1AR action in human physiology, we characterized the pharmacologic properties of the human A1AR, examined somatic sites of A1AR gene expression, and identified the chromosomal location of the human A1AR gene. Using stably transfected CHO cells, the ligand binding properties of human and rat A1ARs were directly compared. Saturation studies showed that the human and rat A1ARs had similar high affinity for the A1 agonist [(3)H]CCPA (human, K(d)=517±64 pM; B(max) 438±29 fmol/mg of protein; rat, K(d)=429±69 pM; B(max) 358±76 fmol/mg of protein). Competition studies performed using seven adenosine agonists and four adenosine antagonists also did not detect differences in the ligand binding properties among the rat and human A1ARs. Northern analysis of 16 human tissues revealed the presence of a single hybridizing transcript of 2.5 kb. Human A1AR receptor mRNA expression was greatest in brain and testis; lower levels of A1AR mRNA were present in heart, pancreas, kidney and spleen. Southern blotting and PCR analysis of human-rodent somatic cell hybrids showed that the A1AR gene is on human chromosome 1. Using fluorescence in situ hybridization, the human A1AR gene was further localized to the 1q32.1 region. These observations show that the human A1AR is a high affinity receptor that has ligand binding properties similar to the rat A1AR, human A1AR mRNA is heavily expressed in brain and testis, and the gene encoding the human A1AR is present on the long arm of chromosome 1.