Cloning of human immunoglobulin mu gene and comparison with mouse mu gene

We have cloned a 12 kb DNA segment containing human mu gene and its flanking sequence from human fetal liver DNA library using mouse mu gene as a probe. Partial nucleotide sequence determination shows that the cloned DNA contains the sequence encoding human mu chain. This is the first constant region gene of the human heavy chain that is cloned. We have compared human and mouse mu genes by heteroduplex analysis and Southern blot hybridization. The results clearly show that not only the sequence encoding the CH4 domain but also the 5'-flanking (S mu) sequence is conserved between human and mouse mu genes, suggesting that the nucleotide sequence in the S mu region has an important biological function, presumably a recognition signal for the class switch recombinant as proposed previously.     

Short-range linkage relationships of the valyl-tRNA synthetase gene in Fugu rubripes

 The class III region of the human major histocompatibility complex (MHC) is gene-dense, averaging one gene every 10–20 kilobases (kb). Its gene order has been compared with other organisms. To extend this analysis further in another non-mammalian vertebrate, the compact genome of Fugu rubripes was investigated for the existence of orthologues of these class III genes and their relative arrangements. Orthologues of the M _r 70000 heat shock protein (HSP70) and valyl-tRNA synthetase genes have been isolated. They do not seem to be closely physically linked as compared with mammals (supported by longer-range analysis using pulsed field gel electrophoresis). Random shotgun sequencing of the two Fugu cosmids containing the gene encoding valyl-tRNA synthetase revealed sequences resembling genes encoding tenascin-X, the nuclear antigen A/Ro of Sjogren’s syndrome, and the Landsteiner-Wiener blood group glycoprotein. These linkage relationships recapitulate some mammalian data, albeit imperfectly. Tenascin-X has been located both in the human and mouse Mhc class III regions. Three copies of a sequence found in the gene encoding Sjogren’s syndrome nuclear antigen A/Ro have been identified in the human Mhc class I region; the mouse Mhc class I region contains one copy. It is postulated that a fragmented gene pattern had existed prior to convergence in the ancestral mammalian immune response-related Mhc region, and that some of these genes had belonged to the same linkage group. Received: 17 February 1997 / Revised: 25 March 1997     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies. Received: 8 July 1997 / Accepted 11 August 1997     

βLCR对β地中海贫血基因在转基因小鼠体内表达的影响

目的:明确在转基因小鼠体内,βLCR对β地中海贫血基因表达的影响。方法：将完整人β-IVSⅡ-654地中海贫血基因,与串连了人βLCR的β-IVSⅡ-654地中海贫血基因分别经显微注射法制作转基因小鼠;荧光定量RT-PCR法检测β-IVSⅡ-654地贫基因在转基因小鼠体内的表达;采用统计分析比较2类转基因鼠中外源基因的表达量。结果：成功建立2类整合了人β-IVSⅡ-654地贫基因的转基因小鼠模型。荧光定量RT-PCR分析结果表明,在整合了串连人βLCR的β-IVSⅡ-654地贫基因的小鼠体内,外源基因mRNA的表达量远高于仅整合β-IVSⅡ-654地贫基因的小鼠（统计分析P值 ）。结论：βLCR核心片段的存在可以使β-珠蛋白基因家族（包括β-地贫基因）在转基因小鼠体内获得高效表达的必要条件。     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

Comprehensive analysis of keratin gene clusters in humans and rodents

Here, we present the comparative analysis of the two keratin (K) gene clusters in the genomes of man, mouse and rat. Overall, there is a remarkable but not perfect synteny among the clusters of the three mammalian species. The human type I keratin gene cluster consists of 27 genes and 4 pseudogenes, all in the same orientation. It is interrupted by a domain of multiple genes encoding keratin-associated proteins (KAPs). Cytokeratin, hair and inner root sheath keratin genes are grouped together in small subclusters, indicating that evolution occurred by duplication events. At the end of the rodent type I gene cluster, a novel gene related to K14 and K17 was identified, which is converted to a pseudogene in humans. The human type II cluster consists of 27 genes and 5 pseudogenes, most of which are arranged in the same orientation. Of the 26 type II murine keratin genes now known, the expression of two new genes was identified by RT-PCR. Kb20, the first gene in the cluster, was detected in lung tissue. Kb39, a new ortholog of K1, is expressed in certain stratified epithelia. It represents a candidate gene for those hyperkeratotic skin syndromes in which no K1 mutations were identified so far. Most remarkably, the human K3 gene which causes Meesmann's corneal dystrophy when mutated, lacks a counterpart in the mouse genome. While the human genome has 138 pseudogenes related to K8 and K18, the mouse and rat genomes contain only 4 and 6 such pseudogenes. Our results also provide the basis for a unified keratin nomenclature and for future functional studies.     

A mapping and evolutionary study of porcine sex chromosome gene

A combination of FISH and RH mapping was used to study the evolution of sex chromosome genes in the pig. In total, 19 genes were identified, including 3 PAR genes (STS, KAL, PRK). The gene order of the porcine X Chromosome (Chr) closely resembled the human X Chr (PRK/STS/KAL–AMELX–EIF2s3X/ZFX–USP9X–DBX–SMCX), suggesting that the porcine X has undergone very little rearrangement during evolution. For the porcine Y Chr, two linkage groups of 10 NRY genes were found, and the following order was established: Ypter–(AMELY–EIF2S3Y/ZFY–USP9Y–DBY/UTY)–(TSPY–SMCY–UBE1Y–SRY)–CEN. This gene order showed greater conservation with the murine Y than with the human Y Chr. In addition, all porcine Y Chr genes mapped to Yp, which is similar to the mouse and included EIF2s3Y and UBE1Y, which are not present in humans. Interestingly, complete conservation of X/Y homologous gene order was found between the pig X and Y Chrs, indicating that the porcine Y Chr has not undergone extensive reorganisation with respect to the X. This suggests that the order of the X/Y homologous genes of the porcine X and Y Chrs may closely resemble the ancestral gene order of the eutherian sex chromosomes.     

A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors

Summary A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to theEscherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-β cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 μM). Such clones expressed an increased level of TGF-β when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-β in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.     

离子通道亚型与其基因共表达的关联研究

离子通道亚型与其基因共表达的关联对研究离子通道功能有重要意义。文章采用主成分分析和模糊C-均值聚类算法对数据进行分析, 将方法应用到人类和小鼠两套表达谱数据, 结果发现离子通道亚型中钾离子通道、钙离子通道、氯离子通道和受体激活型离子通道的表达谱聚类结果与生物学分类有较好的一致性, 体现了离子通道亚型在mRNA水平上的共表达, 并证实了通过离子通道表达谱能很好的对离子通道的功能亚型进行分类。     

Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z

We isolated and characterized the gene encoding human transglutaminase (TG)(X) (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG(Z)) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG(X) and TG(Z) showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TG(C),TG(E), and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.     

Comparative genomic sequencing reveals a strikingly similar architecture of a conserved syntenic region on human chromosome 11p15.3 (including gene ST5) and mouse chromosome 7

Comparative genomics is a superior way to identify phylogenetically conserved features like genes or regions involved in gene regulation. The comparison of extended orthologous chromosomal regions should also reveal other characteristic traits essential for chromosome or gene function. In the present study we have sequenced and compared a region of conserved synteny from human chromosome 11p15.3 and mouse chromosome 7. In human, this region is known to contain several genes involved in the development of various disorders like Beckwith-Wiedemann overgrowth syndrome and other tumor diseases. Furthermore, in the neighboring chromosome region 11p15.5 extensive imprinting of genes has been reported which might extend to region 11p15.3. The analysis of approximately 730 kb in human and 620 kb in mouse led to the identification of eleven genes. All putative genes found in the mouse DNA were also present in the same order and orientation in the human chromosome. However, in the human DNA one putative gene of unknown function could be identified which is not present in the orthologous position of the mouse chromosome. The sequence similarity between human and mouse is higher in transcribed and exon regions than in non-transcribed segments. Dot plot analysis, however, reveals a surprisingly well-conserved sequence similarity over the entire analyzed region. In particular, the positions of CpG islands, short regions of very high GC content in the 5' region of putative genes, are similar in human and mouse. With respect to base composition, two distinct segments of significantly different GC content exist as well in human as in the mouse. With a GC content of 45% the one segment would correspond to "isochore H1" and the other segment (39% GC in human, 40% GC in mouse) to "isochore L1/L2". The gene density (one gene per 66 kb) is slightly higher than the average calculated for the complete human genome (one gene per 90 kb). The comparison of the number and distribution of repetitive elements shows that the proportion of human DNA made up by interspersed repeats (43.8%) is significantly higher than in the corresponding mouse DNA (30.1%). This partly explains why the human DNA is longer between the landmark genes used to define the orthologous positions in human and mouse.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.     

The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4.

Atrial natriuretic factors (ANF) are polypeptides having natriuretic, diuretic, and smooth muscle-relaxing activities that are synthesized from a single larger precursor: pronatriodilatin. Chromosomal assignment of the gene coding for human pronatriodilatin was accomplished by in situ hybridization of a [3H]-labeled pronatriodilatin probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs with normal and rearranged chromosomes 1. The human pronatriodilatin gene was mapped to the distal short arm of chromosome 1, in band 1p36. Southern blot analysis of mouse X Chinese hamster somatic cell hybrids was used to assign the mouse pronatriodilatin gene to chromosome 4. This assignment adds another locus to the conserved syntenic group of homologous genes located on the distal half of the short arm of human chromosome 1 and on mouse chromosome 4.     

Genomic organization and mapping of the human and mouse neuronal β2-nicotinic acetylcholine receptor genes

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the β2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60–80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human β2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3. Received: 26 January 1999 / Accepted: 10 May 1999