Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Four ubiquitously expressed genes, RD (D6S45)-SKI2W (SKIV2L)-DOM3Z-RP1 (D6S60E), are present between complement component genes factor B and C4 in the class III region of the HLA

The association of the HLA class III region with many diseases motivates the investigation of unidentified genes in the 30-kb segment between complement component genes Bf and C4. RD, which codes for a putative RNA binding protein, is 205 bp downstream of Bf. SKI2W (HGMW-approved symbol SKIV2L), a DEVH-box gene probably involved in RNA turnover, is 171 bp downstream of RD (HGMW-approved symbol D6S45). RP1 (HGMW-approved symbol D6S60E) is located 611 bp upstream of C4. The DNA sequence between human RD and RP1 was determined and the exon-intron structure of SKI2W elucidated. SKI2W consists of 28 exons. The putative RNA helicase domain of Ski2w is encoded by 9 exons. Further analysis of the 2.5-kb intergenic sequence between SKI2W and RP1 led to the discovery of DOM3Z. The full-length cDNA sequence of DOM3Z encodes 396 amino acids with a leucine zipper motif. Dom3z-related proteins are present in simple and complex eukaryotes. In Caenorhabditis elegans, Dom3z-related protein could be involved in the development of germ cells. Human RD-SKI2W and DOM3Z-RP1 are arranged as two head-to-head oriented gene pairs with unmethylated CpG sequences at the common 5' regulatory region of each gene pair. The ubiquitous expression pattern suggests that these four genes are probably housekeeping genes.     

An outbreak of a mixed infection of Dermatophilus congolensis and Microsporum gypseum in camels (Camelus dromedarius) in Saudi Arabia

We isolated and identified the major protein present in corneas with granular dystrophy (GCD). We compared Coomassie-blue-stained protein bands obtained on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the extracts of corneas with GCD, corneas with other disorders, and normal human corneal tissue. After SDS-PAGE and transfer to a polyvinylidene difluoride membrane, bands of interest were analyzed by amino acid sequencing and by Western blotting. Corneas with GCD were also examined immunohistochemically. On SDS-PAGE a 63-kd band just below albumin was present in extracts of all corneas. The albumin/63-kd ratio was normally approximately 3:1, suggesting that the protein is a dominant constituent of the cornea. This band was much more plentiful than normal in corneas with GCD. Amino-terminal sequence analysis of the protein revealed a Gly-Pro-Ala-Lys-Ser-Pro-Tyr-Gln-Leu-Val-Leu-Gln-His-Ser-Arg sequence indistinguishable from an amino-terminal protein sequence deduced from a cDNA clone designated beta ig-h3, and it as well as the abnormal accumulations in GCD cross-reacted with beta ig-h3 antiserum. The presence of excessive beta ig-h3 in human corneas with GCD together with reported mutations in the beta ig-h3 gene in GCD suggests that the mutated gene product is a fundamental constituent of the characteristic corneal accumulations in GCD.     

Gene and locus structure and chromosomal localization of the protease-activated receptor gene family

Protease-activate receptors (PARs) mediate activation of platelets and other cells by thrombin and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis, and other normal and pathological processes. We report here the structure of the mouse and human PAR3 genes as well as the organization of a PAR gene cluster encompassing the genes encoding PARs 1, 2, and 3. We also report the structure of the mouse and human PAR4 genes, which map to distinct chromosomal locations and encode a new thrombin receptor. PARs 1-4 are all encoded by genes with the same two exon structure. In each case, exon 1 encodes a signal peptide, and exon 2 encodes the mature receptor protein. These are separated by an intron of variable size. The genes encoding PARs 1-3 all map to chromosome 13D2 in mouse and chromosome 5q13 in human. In mouse, all three genes are located within 80 kilobases of each other. The PAR1 gene is located centrally and is flanked upstream by the PAR3 gene and downstream by the PAR2 gene in both species. The proximity of the PAR1 and PAR3 genes suggests the possibility that these genes might share regulatory elements. A comparison of the structures of the PAR amino acid sequences, gene structures, locus organization, and chromosomal locations suggests a working model for PAR gene evolution.     

The maternal par genes and the segregation of cell fate specification activities in early Caenorhabditis elegans embryos

After fertilization in C. elegans, activities encoded by the maternally expressed par genes appear to establish cellular and embryonic polarity. Loss-of-function mutations in the par genes disrupt anterior-posterior (a-p) asymmetries in early embryos and result in highly abnormal patterns of cell fate. Little is known about how the early asymmetry defects are related to the cell fate patterning defects in par mutant embryos, or about how the par gene products affect the localization and activities of developmental regulators known to specify the cell fate patterns made by individual blastomeres. Examples of such regulators of blastomere identity include the maternal proteins MEX-3 and GLP-1, expressed at high levels anteriorly, and SKN-1 and PAL-1, expressed at high levels posteriorly in early embryos. To better define par gene functions, we examined the expression patterns of MEX-3, PAL-1 and SKN-1, and we analyzed mex-3, pal-1, skn-1 and glp-1 activities in par mutant embryos. We have found that mutational inactivation of each par gene results in a unique phenotype, but in no case do we observe a complete loss of a-p asymmetry. We conclude that no one par gene is required for all a-p asymmetry and we suggest that, in some cases, the par genes act independently of each other to control cell fate patterning and polarity. Finally, we discuss the implications of our findings for understanding how the initial establishment of polarity in the zygote by the par gene products leads to the proper localization of more specifically acting regulators of blastomere identity.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, from Arabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plant Arabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designated AtUBC1-3 and AtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of the AtUBC1-3 and AtUBC4-6 genes by the histochemical analysis of transgenic Arabidopsis containing the corresponding promoters fused to the beta-glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between the AtUBC1-3 and AtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s in Arabidopsis.     

Ilarviruses encode a Cucumovirus-like 2b gene that is absent in other genera within the Bromoviridae

We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3' end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.     

Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function

Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.