Further evidence for the dispersion of the human fibrillar collagen genes.

Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2.     

A physical map of the human PI and AACT genes

We have used probes from the human genes PI, PIL, and AACT (alpha 1-antitrypsin, alpha 1-antitrypsin-related sequence, and alpha 1-antichymotrypsin) to make a pulsed-field map of the surrounding region of 14q31-32. We have discovered that the PI-PIL gene cluster is only 220 kb away from the AACT gene and that it is orientated in the opposite direction. The comparatively short distance between the genes comes as a surprise given previous estimates of the level of genetic recombination between them.     

Assignment of the alpha 1-antitrypsin gene and a sequence-related gene to human chromosome 14 by molecular hybridization

alpha 1-Antitrypsin is a major plasma protease inhibitor synthesized in the liver. Genetic deficiency of this protein predisposes the affected individuals to development of infantile liver cirrhosis or chronic obstructive pulmonary emphysema. The human chromosomal alpha 1-antitrypsin gene has been cloned and shown to contain three introns in the peptide-coding region. When the cloned alpha 1-antitrypsin gene was used as a hybridization probe to analyze Eco RI-digested genomic DNA from different individuals, two distinct bands of 9.6 kilobases (kb) and 8.5 kb in length were observed in every case. Further analysis using only labeled intronic DNA as the hybridization probe has indicated that the authentic alpha 1-antitrypsin gene resides within the 9.6-kb fragment. Thus the 8.5-kb fragment must contain another gene that is closely related in sequence to the alpha 1-antitrypsin gene. Using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the alpha 1-antitrypsin gene as well as the sequence-related gene have been assigned to human chromosome 14 by Southern hybridization and synteny analysis.     

Mapping of Aldose Reductase Gene Sequences to Human Chromosomes 1, 3, 7, 9, 11, and 13

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, we mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q42, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other active genes, non-aldose reductase homologous sequences, or pseudogenes.     

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.     

Localization of human alpha 1 acid glycoprotein genes to 9q31----34.1

There is evidence for more than one alpha 1 acid glycoprotein (alpha 1 AGP) gene, and they all appear to be in close proximity. In situ hybridization of the cloned human cDNA p alpha 1AGP-2 to human chromosomes indicates that the alpha 1AGP genes are located between bands q31 and q34.1 on chromosome 9. This finding is in agreement with the previous assignment of the locus for alpha 1AGP to a linkage group with ABO and AK on chromosome 9.     

Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.     

A review and comparison of the murine alpha1-antitrypsin and alpha1-antichymotrypsin multigene clusters with the human clade A serpins

The major human plasma protease inhibitors, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, are each encoded by a single gene, whereas in the mouse they are represented by clusters of 5 and 14 genes, respectively. Although there is a high degree of overall sequence similarity within these groupings, the reactive-center loop (RCL) domain, which determines target protease specificity, is markedly divergent. The literature dealing with members of these mouse serine protease inhibitor (serpin) clusters has been complicated by inconsistent nomenclature. Furthermore, some investigators, unaware of the complexity of the family, have failed to distinguish between closely related genes when measuring expression levels or functional activity. We have reviewed the literature dealing with the mouse equivalents of human alpha(1)-antitrypsin and alpha(1)-antichymotrypsin and made use of the recently completed mouse genome sequence to propose a systematic nomenclature. We have also examined the extended mouse clade "a" serpin cluster at chromosome 12F1 and compared it with the syntenic region at human chromosome 14q32. In summarizing the literature and suggesting a standardized nomenclature, we aim to provide a logical structure on which future research may be based.     

Complete cDNA sequence and chromosomal localization of mouse alpha 1-antitrypsin

A cDNA encoding the complete open reading frame of murine alpha 1-antitrypsin has been cloned and sequenced. The nucleic acid and predicted amino acid sequences show homology to human alpha 1-antitrypsin and demonstrate the preservation of critical structural determinants for intracellular targeting, carbohydrate attachment, and catalytic function. The alpha 1-antitrypsin gene locus (Aat) has been localized on murine chromosome 12E----F by in situ hybridization.     

Assignment of the fucosidase pseudogene FUCA1P to chromosome region 2q31----q32

FUCA1P is a pseudogene of the structural fucosidase gene FUCA1. The former has been mapped to human chromosome 2, whereas the latter has been localized to chromosome 1p34----p36. We have further localized FUCA1P to chromosomal band 2q31----q32 by fluorescent in situ hybridization and digital imaging microscopy. This localization was confirmed by linkage analysis between FUCA1P and the COL3A1 gene in 2q24----q32 which gave maximal lod scores of 4.03 at 3% recombination.     

Regional mapping of the human renin gene to 1q32 by in situ hybridization

Renin, related to other aspartyl proteases, plays an important role in the cascade which regulates blood pressure and salt metabolism. A human renin 1 100 bp long cDNA including most of the coding region and the 3' non coding region has been subcloned by Soubrier et al., 1983. A 1000 b RNA probe derived by subcloning into pSP64 vector was hybridized to EcoRI and HindIII digests of the DNA of a panel of 24 man-rodent somatic cell hybrids. With HindIII, four restriction fragments were observed, two of them revealing polymorphism (8.4 kb and 6.0 kb). Analysis of the distribution of the human signal among the hybrids confirms the localization of the renin gene (REN) to human chromosome 1. The whole plasmid including the 1 100 bp long insert was used for regional mapping by in situ hybridization; 45% of silver grains were found on chromosome 1, with a clear peak at band 1q32 (33% of silver grains on chromosome 1) and a smaller one at band 1q42 (17%). These data favour a regional localization of the renin gene to 1q32-1q42. Mac Gill et al. (1987) have localized the REN gene to 1q25-1q32 using in situ hybridization. Thus, 1q32 could be the most probable localization. No other peak could be observed. This is in agreement with results obtained with somatic cell hybrids.     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

Identification and localization of MEP1A-like sequences (MEP1AL1-4) in the human genome.

The human MEP1A gene encodes the meprin alpha subunit that consists of a protease domain conserved in the astacin family of metalloendopeptidases and several C-terminal interaction domains present in other proteins. Using the alpha subunit cDNA, we identified two clones from a human P1-derived artificial chromosome (PAC) library. Fluorescence in situ hybridization (FISH) mapped both PACs (1e12, 65a14) to chromosome 6p21, confirming the MEP1A location. FISH also mapped PAC 65a14 to chromosome 13cen, and to chromosome 9 in three different regions, 9p12-13, 9q21, and 9q22. Southern blot analysis showed that sequences of PAC 65a14 and MEP1A were similar in the 3' end but different in the 5' end, revealing for the first time that the human genome may encode multiple interaction domains highly similar to those of the meprin alpha subunit. The symbols of MEP1AL1, MEP1AL2, MEP1AL3, and MEP1AL4 have been designated for MEP1A-like sequences on 9p12-13, 9q21, 9q22, and 13cen, respectively.     

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.     

Chromosomal localization of the human interleukin 1 alpha (IL-1 alpha) gene

The human interleukin 1 alpha gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12-21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1 alpha gene maps to the same general region on the long arm of chromosome 2 as the IL-1 beta gene, which has been previously assigned.     

Chromosomal localization of leukocyte interferon gene in the pig (Sus scrofa domestica L.) by in situ hybridization

Using as a probe pig genomic DNA, including the complete interferon alpha gene, we have mapped the leukocyte interferon gene on pig chromosome 1 by in situ hybridization. A total of 196 silver grains were noted on the 106 metaphases scored: 31% of the grains were observed on chromosome 1, and 67% of these were localized in the region 1q2.2----q2.7.     

Chromosomal localization of human and rat A alpha, B beta, and gamma fibrinogen genes by in situ hybridization

In situ hybridization of radiolabeled fibrinogen cDNAs to human and rat metaphase chromosomes has shown that the genes encoding the A alpha, B beta, and gamma fibrinogen subunits are syntenic in both species. Our data localize the human fibrinogen gene cluster to band q31 on chromosome 4, thereby confirming and extending previous map assignments of these genes in man. We have also assigned these genes to the q31----q34 region of rat chromosome 2. This is the first map assignment of these genes in the rat and also the first report to clearly establish linkage of the B beta subunit gene to the A alpha and gamma genes in this species.     

用人染色体14q24.3区带探针池直接分离表达顺序

本文报道了从显微切割的人染色体区带直接分离区带专一性表达序列的方法和结果。