Further characterization of a somatic cell hybrid panel: ten new assignments to the bovine genome

Thirty-six partially characterized hamster-bovine hybrid cell lines were used for the determination of synteny groups. Sixteen additional reference loci, selected for their coverage of the bovine genome, were analysed on these hybrid cells. This increases to 25 the number of synteny groups detected. This panel was then used to make synteny assignments for 10 additional loci, eight by Southern blotting (COL1A1, COL1A2, FAS, CTSB, CTSL, CHRNG, HEXB and HTR1A) and two by polymerase chain reaction (PCR) amplification (HRH1 and ETH1112), These loci were assigned to international synteny groups U12 (HRH1), U13 (COL1A2), U17 (CHRNG), U21 (COL1A1, FAS), U29 (ETHI1112), to chromosome 20 (U14 or U25) for HEXB and HTR1A, and to the same local synteny group (A), which is probably U18, for CTSB and CTSL. For three loci already mapped in humans (COL1A1, COL1A2 and CHRNG), the present results are in accordance with the predictions based on comparative mapping between the human and bovine species.     

Assignment of the casein kinase II gene family to cattle chromosomes

Theileriosis, or East Coast fever, a parasitic disease in cattle, is associated with overexpression of casein kinase II. Casein kinase II is composed of two catalytic subunits (α or α') and two regulatory β subunits. The genes encoding these subunits of casein kinase II were mapped to bovine chromosomes by polymerase chain reaction analysis of a well-characterized bovine × rodent somatic hybrid cell panel. The α-subunit (CSNK2A1) was mapped to bovine chromosome 13, the α'-subunit (CSNK2A2) to chromosome 5 and the β-subunit (CSNK2B) to chromosome 23. Both CSNK2A1 and CSNK2B mapped to known regions of conserved synteny between human and cattle, while CSNK2A2 defined a new homology segment between the human and bovine genomes.     

Assignment of bovine synteny group U2 to chromosome 9

One cosmid containing a microsatellite (INRA144, D9S14) was assigned to bovine synteny group U2 by somatic cell genetics and localized to bovine chromosome 9q25 by fluorescent in situ hybridization. These results permitted the assignment of one more synteny group to a bovine chromosome. There are now 22 out of 31 bovine synteny groups which are related to a chromosome. The mapping data have been entered in the BovMap database, Jouy-en-Josas, France.     

Assignment of the gene for dipeptidase 2 to Mus musculus chromosome 18 by somatic cell hybridization

Evidence is presented for the assignment of the gene for dipeptidase 2 to Mus musculus chromosome 18 by synteny testing and karyotypic analysis of Chinese hamster × mouse somatic cell hybrid clones. DIP-2 and chromosome 18 were expressed concordantly in 24/24 clones examined (ten primary clones and 14 secondary clones). Synteny testing indicated that DIP-2 was not expressed concordantly with the expression of any marker enzymes.This work was supported by NIH grant USPHS GM 09966.     

Assignment of the HOX2 and HOX3 gene clusters to the bovine chromosome regions 19q17-qter and 5q14-23

The homeobox 2 (HOX2) and homeobox 3 (HOX3) clusters have been chromosomally assigned in cattle by in situ hybridization. The probes employed were a murine probe for the mapping of HOX2 to 19q17-qter and human probes for the mapping of HOX3 to 5q14-q23. These assignments confirm the chromosomal assignment of two syntenic groups, consisting of loci located on human chromosome 12 (bovine chromosome 5) and the long arm of human chromosome 17 (bovine chromosome 19).     

The bovine fibroblast growth factor receptor 3 (FGFR3) gene is not the locus responsible for bovine chondrodysplastic dwarfism in Japanese brown cattle

Fibroblast growth factor receptor 3 (FGFR3) is one of the four distinct membrane-spanning tyrosine kinase receptors for fibroblast growth factors. The FGFR3 is a negative regulator of endochondral ossification and mutations in the FGFR3 gene have been found in patients of human hereditary diseases with chondrodysplastic phenotypes. Recently, we mapped the locus responsible for hereditary chondrodysplastic dwarfism in Japanese brown cattle to the distal region of bovine chromosome 6 close to the FGFR3 gene, suggesting that FGFR3 was a positional candidate gene for this disorder. In the present study, we isolated complementary DNA (cDNA) clones containing the entire coding region of the bovine FGFR3 gene. Comparison of the nucleotide sequence between affected and normal animals revealed no disease-specific differences in the deduced amino acid sequences. We further refined the localization of FGFR3 by radiation hybrid mapping, which is distinct from that of the disease locus. Therefore we conclude that bovine chondrodysplastic dwarfism in Japanese brown cattle is not caused by mutation in the FGFR3 gene.     

Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes

We described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the "type II" viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.     

Physical mapping of CSF2RA, ANT3 and STS on the pseudoautosomal region of bovine chromosome X

The pseudoautosomal region (PAR) of bovine chromosome X (BTA X) has a particularly low representation of genes and markers, making comparative gene mapping in this region difficult. We describe the localization of three genes, colony-stimulating factor 2 receptor alpha (CSF2RA), ADP/ATP translocase 3 (ANT3) and steroid sulphatase (STS) on PAR of BTA X using a 5000 rad whole-genome radiation hybrid panel. The relationship of these genes to a number of previously mapped simple sequence repeat (microsatellite) markers is determined by physical and radiation hybrid mapping methods. The resulting radiation hybrid map resolves a discrepancy between the two major bovine linkage maps in the PAR of BTA X.     

一个有假基因特点的鼻咽癌相关基因的分离和克隆

用定位候选克隆法克隆了一个与鼻咽癌相关的基因,命名为NAG73基因,结构分析显示NAG73的基因序列无内含子,无典型的启动子序列,polyA尾,与人类生长激素促分泌因子基因的第4个外显子和3′端非翻译区同源,说明NAG73可能是人类生长激素促分泌因子基因的假基因,同时,实验证明NAG73在一些细胞和组织中活跃表达,在正常鼻咽上皮细胞,鼻咽癌上皮细胞和鼻咽癌活检组织,NAG73有表达差异,序列分析显示NAG73有编码翻译产物的可能,NAG73可能可编码产物,该产物在鼻咽癌的发病发展中发挥作用。     

The porcine proteasome subunit A4 (PSMA4) gene: isolation of a partial cDNA, linkage and physical mapping

A partial cDNA clone encoding the porcine proteasome subunit A4 ( PSMA4 or proteasome subunit C9) has been isolated from a porcine muscle cDNA library and sequenced. A biallelic Taq I RFLP was identified in Large White, Landrace and Duroc breeds. Moreover, the 3'-untranslated region of the gene showed a triallelic SSCP. By linkage analysis the PSMA4 locus was assigned to pig chromosome 7 and by radioactive in situ hybridization this locus was mapped to the region 7q13–q14.     

对家蚕第18连锁群隐性基因elp、ch-2和mln测交系的分子定位分析

家蚕长形卵(elp)、第二隐性赤蚁(ch-2)、暗化型(mln)均为第18染色体上的隐性突变, 在经典连锁图谱上的顺序和遗传距离已经排定。文章采用正常卵、正常黑蚁及正常白蛾品种P50与包含此3个隐性突变的三隐性测交系W18组配正反交群体, F1回交W18后获得回交群体(P50×W18)♀×W18♂ 和W18♀×(P50×W18)♂, 分别记作BC1F和BC1M, 利用已构建的家蚕SSR分子连锁图谱和根据家蚕基因组精细图设计的STS标记, 对这3个突变基因elp、ch-2、mln进行了分子定位研究, 并根据家蚕基因组精细图, 将第18连锁群的经典遗传图、分子连锁图和基因组物理图进行了对应。整合后的图谱遗传距离为94.2 cM, 突变基因和分子标记的排列顺序分别与形态标记连锁图和基因组精细图相一致, 研究结果对家蚕第18 染色体上其他突变的定位与克隆有重要的借鉴作用。     

A newly synthesized selective casein kinase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification of casein kinase I from bovine testis

When screening various isoquinolinesulfonamide compounds which we synthesized, CKI-7, N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, was found to have a potent inhibitory action against casein kinase I and a much weaker effect on casein kinase II and other protein kinases. Kinetic analysis indicated that CKI-7 inhibited casein kinase I competitively with respect to ATP and that the Ki values were 8.5 microM for casein kinase I and 70 microM for casein kinase II. An affinity chromatography absorbent was synthesized by coupling CKI-8 (1-(5-chloroisoquinoline-8-sulfonyl], a derivative of CKI-7, to cyanogen bromide-activated Sepharose 4B. Partially purified casein kinase I from bovine testis was subjected to affinity chromatography. Analysis of the purified casein kinase I by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single band with molecular weight 37,000. These newly synthesized compounds, CKI-7 and CKI-8, should serve as useful tools for elucidating the biological significance of casein kinase I-mediated reactions.     

Mapping of the mouse bilirubin UDP-glucuronosyltransferase gene (Gnt-1) to chromosome 1 by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLVs) were detected by using a rat cDNA probe for the bilirubin UDP-glucuronosyltransferase (UDPGT) gene between two mouse strains, 129/Sv and MOL-MIT. RFLVs of the gene were found byEcoRI andPvuII digestions. From linkage analyses of the three-point cross test usingElo andEn-1 as marker genes, the bilirubin UDPGT gene was mapped at position 37 on chromosome 1. Bilirubin and phenol UDPGTs have been suggested to be expressed by a single gene by alternative splicing in human and rat. The mouse bilirubin UDPGT gene was namedGnt-1.This study was supported by Grant-in-Aid for Research Project A-II from the Institute for Developmental Research, Aichi Prefecture Colony.     

Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus)

This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceeded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study. Received: 6 September 2000 / Accepted: 7 March 2001     

Purification and characterization of the messenger ribonucleoprotein-associated casein kinase II of Artemia salina cryptobiotic gastrulae

The mRNP-associated protein kinase is purified to near homogeneity by ion-exchange chromatography on phosphocellulose and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The cyclic nucleotide-independent enzyme phosphorylates casein using either ATP or GTP. The enzyme exists in two forms composed of subunits with M_r 36 500 (α) and 28 000 (β) and of subunits with M_r 36 500 (α), 33 000 (α′) and 28 000 (β). The undegraded enzyme has an M_r of 136 000 ± 7000. The enzyme is inhibited by heparin and hemin and stimulated by spermine. The mRNP-associated protein kinase may be classified as a casein kinase II. Main mRNP protein phosphate acceptors have M_r values of 112 000, 72 000, 65 000, 53 000, 38 000, 28 000, 23 500 and 21 000. Phosphorylation of the M_r 38 000 poly(A)-binding protein resulted in the generation of different acidic ionic species. From the observed inhibition of the translational activity after phosphorylation by the mRNP-associated protein kinase a function in the repression of mRNP is proposed.     

A dual role for receptor-interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy

Autophagy is triggered by the intracellular bacterial sensor NOD2 (nucleotide-binding, oligomerization domain 2) as an anti-bacterial response. Defects in autophagy have been implicated in Crohn's disease susceptibility. The molecular mechanisms of activation and regulation of this process by NOD2 are not well understood, with recent studies reporting conflicting requirements for RIP2 (receptor-interacting protein kinase 2) in autophagy induction. We examined the requirement of NOD2 signaling mediated by RIP2 for anti-bacterial autophagy induction and clearance of Salmonella typhimurium in the intestinal epithelial cell line HCT116. Our data demonstrate that NOD2 stimulates autophagy in a process dependent on RIP2 tyrosine kinase activity. Autophagy induction requires the activity of the mitogen-activated protein kinases MEKK4 and p38 but is independent of NFκB signaling. Activation of autophagy was inhibited by a PP2A phosphatase complex, which interacts with both NOD2 and RIP2. PP2A phosphatase activity inhibited NOD2-dependent autophagy but not activation of NFκB or p38. Upon stimulation of NOD2, the phosphatase activity of the PP2A complex is inhibited through tyrosine phosphorylation of the catalytic subunit in a process dependent on RIP2 activity. These findings demonstrate that RIP2 tyrosine kinase activity is not only required for NOD2-dependent autophagy but plays a dual role in this process. RIP2 both sends a positive autophagy signal through activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A.