Characterization of Caenorhabditis elegans homologs of the Down syndrome candidate gene DYRK1A

The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.     

A candidate gene for human U1 RNA.

Clones containing sequences complementary to the small nuclear RNA U1 were isolated from the human DNA library of Lawn et al. (1978). Three clones were studied by hybridization and restriction enzyme cleavage. The results showed that the inserts in all three clones were different and that each clone contains one single copy of a sequence which hybridizes to U1 RNA. The results revealed moreover that only one of the three clones contains all the cleavage sites which can be predicted from the known sequence of human U1 RNA, suggesting that the three clones comprise one candidate U1 gene and two pseudogenes. A fragment from the recombinant with the candidate U1 gene was subcloned in the pPR322 plasmid and part of its sequence was determined. The results showed that the subclone contains a sequence which matches that of the human U1 RNA perfectly. The sequence "TATAT" which often is found adjacent to RNA polymerase II start sites, was identified 33-37 base pairs upstream from the beginning of the U1 sequence. Two ten base pairs long, nearly perfect, direct repeats were also identified in the vicinity of the U1 sequence and an imperfect inverted repeat follows immediately after the U1 gene.     

Characterization of bovine TGM1 and exclusion as candidate gene for ichthyosis in Chianina

Ichthyosis is a heterogeneous group of keratinization disorders reported both in human and animals. Two rare, inherited forms have been reported in cattle, both characterized by autosomal recessive transmission. Because mutations of transglutaminase 1 (TGM1) gene are associated with autosomal recessive ichthyosis in people, this gene was investigated as a candidate for the diseases in cattle. Three different polymorphisms were identified in 5' end region of cattle TGM1. Marker homozygosity was not found among affected calves. Linkage analysis excluded (logarithmic odds [LOD] score -2.0) TGM1 as the cause for ichthyosis phenotype in the analyzed Chianina cases.     

Characterization of porcine autism susceptibility candidate 2 as a candidate gene for the number of corpora lutea in pigs

In a previous study, we mapped two quantitative trait loci (QTL) approximately 50cM apart, both influencing the number of corpora lutea in pigs on chromosome 3. One locus included functional candidate genes for proteins related to specific aspects of fertility, such as the follicle-stimulating hormone receptor and the luteinizing hormone/choriogonadotropin receptor. However, specific genes related to the second locus have not yet been identified. This study aims to identify another candidate gene influencing the number of corpora lutea in pigs. Using 12 polymorphic markers, we fine-mapped a narrow region of pig chromosome 3 that had been shown to contain a QTL for corpora lutea. In the critical region, only 1 gene, autism susceptibility candidate 2 (AUTS2), was identified as a positional candidate. Our results demonstrate that the porcine AUTS2 gene consists of 19 exons with a complete open reading frame of 3768bp encoding an AUTS2 protein of 1256 amino acids. We screened the whole coding sequence and parts of the untranslated region for polymorphisms in an F(2) population of Duroc×Meishan crosses. We found 1 ins/del and 7 single nucleotide polymorphisms (SNP), including 2 nonsynonymous variants, c.943C>T in exon 7 and c.2828C>T in exon 19, resulting in P315S and A943V, respectively. The SNP c.943C>T within a proline-rich domain was genotyped in several breeds; the C allele occurred in all breeds, whereas the T allele occurred only in Meishan pigs. Using in situ hybridization, the mRNA expression of the AUTS2 gene was observed on granulosa cells in the porcine ovary and thus may be associated with hormone sensitivity.     

STK25 is a candidate gene for pseudopseudohypoparathyroidism.

We determined the chromosomal location of the mouse gene Stk25, encoding a member of the Ste20/PAK family of serine/threonine kinases, by interspecific backcross analysis. We mapped Stk25 to the central region of mouse chromosome 1 linked to Chrng (formerly Acrg) and En1. This central region of mouse chromosome 1 shares a region of homology with the long arm of human chromosome 2, suggesting that the human homologue of Stk25 would also map to 2q. We proved this prediction of syntenic homology correct by mapping human STK25 to 2q37. Deletion of the 2q37 region has been implicated in the expression of pseudopseudohypoparathyroidism (PPHP), a disease which shares features of the Albright hereditary osteodystrophy (AHO) phenotype. To investigate a pathogenetic relationship between STK25 and PPHP, we carried out fluorescence in situ hybridization (FISH) using an STK25 gene probe and chromosomes from PPHP patients characterized as having small deletions near the distal end of 2q. PPHP patient DNA showed no hybridization to STK25 genomic DNA, indicating that STK25 is contained within the deleted chromosomal region. This finding, in conjunction with previous studies demonstrating the role of Ste20/PAK kinases in heterotrimeric G protein signaling, suggests that STK25 is a positional candidate gene for PPHP.     

Characterization and fed-batch culture of hybridoma overexpressing apoptosis suppressing gene bcl-2

Mouse hybridoma 2E3 transfected with human bcl-2 gene survived longer with increasing expression level of bcl-2 when cultured in DME medium supplemented with 9% serum. One of the transfectants, 2E3BCMGbcl-2, overexpressed bcl-2 and could maintain viable cell density higher than the initial density for more than four days at a low 0.5% serum concentration. In comparison a mock transfectant 2E3BCMG remained viable for only one day. However, both hybridomas died out within a day in serum-free medium. These results suggested that bcl-2 needed a small amount of some serum components to suppress apoptosis of the hybridoma. Overexpression of bcl-2 also suppressed apoptosis of the hybridoma induced by glutamine deprivation. When hybridoma 2E3BCMGbcl-2 was inoculated in DME medium supplemented with 9% serum and cultured for 10 d with additional 2% serum feed at day 4 of the culture, viable cell density increased 2-fold and antibody produced 3-fold, in comparison with mock transfected 2E3 cultured in the same manner. The mock transfectant with additional feed of serum at day 4 of the culture showed no difference in viable cell density and antibody production. These results suggested that the mock transfectant committed to apoptosis before day 4 of the culture and the additional serum at day 4 could not reverse the commitment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the murine Icam-1 gene.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell adhesion molecule that interacts with the leukocyte beta 2 integrins, LFA-1 and Mac-1. Murine inflammatory models are being utilized increasingly to define the role that ICAM-1 induction plays in the initiation of inflammation. We have isolated murine genomic clones that contain the Icam-1 gene including over 2 kb of 5' flanking sequence. The gene for murine Icam-1 spans over 13 kb and is composed of seven exons and six introns. Each of the extracellular immunoglobulin-like domains of ICAM-1 is encoded by a single exon that ends with the first base of the next codon. Examination of ICAM-1 expression in vivo shows that mRNA levels of ICAM-1 are low in all organs except for the lung but increase markedly in multiple organs at 3 h after administration of endotoxin. The 5' flanking region of the murine gene contains a putative TATA box and potential SP-1, AP-1, and AP-3 sites in positions nearly identical to those in the human gene.     

Characterization of a vaccinia-derived recombinant HIV-1 gp160 candidate vaccine and its immunogenicity in chimpanzees.

The human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 was produced in large-scale microcarrier cultures of Vero cells, using a system involving coinfection with two recombinant vaccinia viruses. The immunogenicity of this material was studied in conjunction with a number of different adjuvant formulations, and chimpanzees were then immunized with gp160 in conjunction with Al(OH)3, Al(OH)3 and sodium deoxycholate, and a lipid-based adjuvant. The Al(OH)3-gp160 vaccine formulation elicited very poor immune responses in two chimpanzees, and these animals were further immunized with gp160 in conjunction with a lipid-based adjuvant. Immunization with the latter formulation lead to induction of high-titer neutralizing antibodies, and, following challenge with HIV-1, one chimpanzee demonstrated no evidence of virus infection over a period of 3 years. The second chimpanzee, which had previously been infected with non-A, non-B hepatitis, and two animals immunized with gp160 with Al(OH)3 and deoxycholate were not protected against challenge.     

The axon guidance receptor gene ROBO1 is a candidate gene for developmental dyslexia

Dyslexia, or specific reading disability, is the most common learning disorder with a complex, partially genetic basis, but its biochemical mechanisms remain poorly understood. A locus on Chromosome 3, DYX5, has been linked to dyslexia in one large family and speech-sound disorder in a subset of small families. We found that the axon guidance receptor gene ROBO1, orthologous to the Drosophila roundabout gene, is disrupted by a chromosome translocation in a dyslexic individual. In a large pedigree with 21 dyslexic individuals genetically linked to a specific haplotype of ROBO1 (not found in any other chromosomes in our samples), the expression of ROBO1 from this haplotype was absent or attenuated in affected individuals. Sequencing of ROBO1 in apes revealed multiple coding differences, and the selection pressure was significantly different between the human, chimpanzee, and gorilla branch as compared to orangutan. We also identified novel exons and splice variants of ROBO1 that may explain the apparent phenotypic differences between human and mouse in heterozygous loss of ROBO1. We conclude that dyslexia may be caused by partial haplo-insufficiency for ROBO1 in rare families. Thus, our data suggest that a slight disturbance in neuronal axon crossing across the midline between brain hemispheres, dendrite guidance, or another function of ROBO1 may manifest as a specific reading disability in humans.     

抑癌基因D L C-1 的研究进展

DLC-1（frequently deletedin liver cancer）基因是新发现的一个肿瘤抑制基因。它的失活有可能参与肿瘤的发生和发展。本文拟就DLC-1基因的结构功能及其在遗传和表遗传方面的失活机制作一综述。     

A candidate gene for human U1 RNA

[This corrects the article on p. 133 in vol. 1, PMID: 6201353.].     

Organization of the human synphilin-1 gene, a candidate for Parkinson's disease

We have recently identified a protein we called synphilin-1, which interacts in vivo with alpha-synuclein. Mutations in alpha-synuclein cause familial Parkinson's disease (PD). Alpha-synuclein protein is present in the pathologic lesions of familial and sporadic PD, and diffuse Lewy body disease, indicating an important pathogenic role for alpha-synuclein. Here we describe the structure of the human synphilin-1 gene (SNCAIP). The open reading frame of this gene is contained within ten exons. We have designed primers to amplify each SNCAIP exon, so these primers can now be used to screen for mutations or polymorphisms in patients with Parkinson's disease or related diseases. We found a highly polymorphic GT repeat within intron 5 of SNCAIP, suitable for linkage analysis of families with PD. We have mapped SNCAIP locus to Chromosome (Chr) 5q23.1-23.3 near markers WI-4673 and AFMB352XH5. In addition, using immunohistochemistry in human postmortem brain tissue, we found that synphilin-1 protein is present in neuropil, similar to alpha-synuclein protein. Because of its association with alpha-synuclein, synphilin-1 may be a candidate for involvement in Parkinson's disease or other related disorders. Received: 21 September 1999 / Accepted: 16 March 2000     

Characterization of a candidate Trypanosoma rangeli small nucleolar RNA gene and its application in a PCR-based parasite detection

In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.     

NECC1, a candidate choriocarcinoma suppressor gene that encodes a homeodomain consensus motif

We isolated a candidate choriocarcinoma suppressor gene from a PCR-based subtracted fragmentary cDNA library between normal placental villi and the choriocarcinoma cell line CC1. This gene comprises an open reading frame of 219 nt encoding 73 amino acids and contains a homeodomain as a consensus motif. This gene, designated NECC1 (not expressed in choriocarcinoma clone 1), is located on human chromosome 4q11-q12. NECC1 expression is ubiquitous in the brain, placenta, lung, smooth muscle, uterus, bladder, kidney, and spleen. Normal placental villi expressed NECC1, but all choriocarcinoma cell lines examined and most of the surgically removed choriocarcinoma tissue samples failed to express it. We transfected this gene into choriocarcinoma cell lines and observed remarkable alterations in cell morphology and suppression of in vivo tumorigenesis. Induction of CSH1 (chorionic somatomammotropin hormone 1) by NECC1 expression suggested differentiation of choriocarcinoma cells to syncytiotrophoblasts. Our results suggest that loss of NECC1 expression is involved in malignant conversion of placental trophoblasts.