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。     

Assignment of human porphobilinogen deaminase to 11q24.1----q24.2 by in situ hybridization and gene dosage studies

In situ hybridization and gene dosage-effect studies were conducted to determine the detailed chromosomal location of the gene encoding human porphobilinogen deaminase (PBGD). Red cell PBGD activity was normal in one patient with monosomy for 11q24.2----qter but was increased 1.5 times in another patient with trisomy for 11q22.2----qter. The cDNA probe for PBGD was found to be specifically hybridized to band 11q24. These results suggest that the gene for PBGD is localized within the region 11q24.1----q24.2.     

The gene for clotting factor 10 is mapped to 13q32----qter

The structural gene for the human clotting factor 10 (F10) has been mapped to chromosome 13 with a cDNA probe hybridized to DNAs from a panel of human X hamster hybrids. In situ hybridization was used to assign F10 to region 13q32----qter of chromosomes from normal human lymphocytes.     

Localization of the genes for histatins to human chromosome 4q13 and tissue distribution of the mRNAs.

A cDNA coding for histatin 1 was isolated from a human submandibular-gland library and sequenced. This cDNA was used to probe RNAs isolated from a variety of tissues to investigate tissue-specific regulation and to determine whether histatins might play a role other than in the oral cavity. The same probe was also used for Southern blot analysis of human genomic DNA restricted with various enzymes, and it showed that the genes coding for histatins are on the same chromosome. In situ hybridization of the cDNA probe to metaphase chromosome spreads was performed to determine chromosomal location of the genes for histatins. A genomic fragment isolated using the cDNA probe was also hybridized to chromosome spreads, and the same chromosome was identified. The genes for histatins are located on chromosome 4, band q13. We have shown that three histatin mRNAs are expressed in human parotid and submandibular glands but in none of the other tissues studied. These results suggest that histatins are specific to salivary secretions.     

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.     

Different chromosomal localization of two adenylyl cyclase genes expressed in human brain

Summary Recently, we characterized a cDNA clone that encodes a human brain adenylyl cyclase (HBAC1). In the present study, we identified a second population of mRNA suspected to encode a new brain adenylyl cyclase (HBAC2). The amino acid sequence of HBAC2 displays significant homology with HBAC1 in the highly conserved adenylyl cyclase domain (250 aminio acids), found in the 3 cytoplasmic domain of all mammalian adenylyl cyclases. However, outside this domain, the homology is extremely low, suggesting that the corresponding mRNA originates from a different gene. We report here the first chromosomal localization of the adenylyl cyclase genes determined by in situ hybridization of human metaphase chromosomal spreads using human brain cDNA probes specific for each mRNA. The probe corresponding to HBAC1 exhibited a strong specific signal on chromosome 8q24, with a major peak in the band q24.2. In contrast, the HBAC2 probe hybridized to chromosome 5p15, with a major peak in the band p15.3. The two cDNAs hybridized at the two loci without any cross reactivity. Thus, in human brain, a heterogeneous population of adenylyl cyclase mRNAs is expressed, and the corresponding genes might be under the control of independent regulatory mechanisms.Abbreviations C catalytic part of adenylyl cyclase - BBAC bovine brain - HBAC human brain - ROAC rat olfactory - RLAC rat liver - RTAC rat testis adenylyl cyclase - G guanine nucleotide GTP binding protein (s, stimulatory; i, inhibitory)     

Localization of the human gene for the El alpha subunit of branched chain keto acid dehydrogenase (BCKDHA) to chromosome 19q13.1----q13.2

The gene encoding the El alpha subunit of branched chain keto acid dehydrogenase (BCKDHA) was mapped to human chromosome region 19q13.1----q13.2 using 3H-labeled cDNA hybridized in situ to human chromosomes.     

Molecular analysis of overlapping chromosomal deletions in patients with Langer-Giedion syndrome.

We have obtained lymphoblastoid cell lines from three patients with Langer-Giedion syndrome who have overlapping deletions in 8q24.1. To isolate the deletion chromosomes from their normal homologs, patient cell lines were fused with hamster cells and hybrid cells were selected for retention of human chromosome 8. These hybrid cell lines were screened for the presence of chromosome 8 by fluorescence in situ hybridization and by Southern blot hybridization. We have hybridized 31 recombinant DNA clones derived from the 8q22-qter region to Southern blots of the hybrid cell lines; 8 were found to lie within the deletion of at least one patient. One clone identified sequences that were missing from one copy of chromosome 8 in all three patients. These clones help to further define the deletions in these patients and will serve as starting points for detailed characterization of the region.     

Molecular characterization of a cDNA encoding functional human CLK4 kinase and localization to chromosome 5q35 [correction of 4q35

Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 5 [corrected].     

Assignment of the human protein C gene (PROC) to chromosome region 2q14----q21 by in situ hybridization

The protein C gene (PROC) was mapped by in situ hybridization. A genomic DNA probe containing the first three exons was 3H-labeled by nick translation, and this was then hybridized in situ to human chromosome preparations. The results localize the gene to 2q14----q21.     

Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24.

Human mast cell growth factor (MGF) complementary DNAs (cDNAs) were cloned from HeLa cells using the polymerase chain reaction with oligonucleotides corresponding to murine and human MGF sequences. Sequencing of the cloned human MGF polymerase chain reaction products revealed two types of cDNA: a full length form corresponding in size to the murine cDNA, and an alternately spliced clone with a deletion of the sixth exon of the gene. Since membrane-bound MGF is predicted to be proteolytically cleaved within the sequences encoded by exon 6 to generate a soluble protein, this alternately spliced cDNA would likely encode a noncleavable, membrane-bound form of MGF. No difference in biological activity on human bone marrow cells was observed with recombinant, soluble forms of both types of human MGF protein. Our previous localization of the murine MGF gene to the Sl locus on chromosome 10 suggested (via conserved linkage groups) that the human MGF gene would be located on human chromosome 12. Therefore, rodent-human somatic cell hybrids with or without an entire human chromosome 12 and hybrids retaining partial 12 were tested by Southern blot analysis and used to show the presence of the human Mgf locus at chromosome region 12q. Chromosomal in situ hybridization localized the gene to 12q22-q24 in the region predicted by the comparative mapping of the murine Mgf/Sl locus.     

Chromosome mapping of the human RAS-related RAP1A, RAP1B, and RAP2 genes to chromosomes 1p12----p13, 12q14, and 13q34, respectively

Three human cDNAs encoding new RAS-related cDNAs, designated RAP1A, RAP1B, and RAP2, have been isolated previously. The encoded proteins are highly related to RAS in the effector region and share an overall identity with RAS of approximately 50%. Using the complete cDNAs or parts thereof as probes, each RAP gene has been localized on human chromosomes by in situ hybridization. The three genes RAP1A, RAP1B, and RAP2 have been assigned to chromosome bands 1p12----p13, 12q14, and 13q34, respectively.     

Chromosome-band-specific painting: chromosome in situ suppression hybridization using PCR products from a microdissected chromosome band as a probe pool

Summary We describe a chromosome-band-specific painting method that involves (1) microdissection of the chromosome, chromosomal region or band, (2) amplification of a variety of chromosome/region/band-specific DNA fragments with the polymerase chain reaction (PCR), and (3) chromosome in situ suppression hybridization (CISS) with the direct use of the PCR products as a probe pool. With this method, it was possible 1) to paint an entire X or Y chromosome, a distal one-fourth of 2q, and only a band at 8q24.1, 2) to identify the origin of a minute marker chromosome in a mentally retarded patient, 3) to detect an X;Y translocation in another patient, and 4) to identify one human chromosome 2 in a human-mouse hybrid cell line. This method allows us to identify not only structural chromosome abnormalities at the band level, but also the origin of cytogenetically unidentifiable marker chromosomes. It will also be useful in studies of evolutionary cytogenetics.     

Chromosomal banding patterns in human large bowel adenomas

Summary The human thyroglobulin gene was mapped by in situ hybridization whereby a ³H-labeled recombinant plasmid DNA containing a fragment of 2.3 kilobases of human thyroglobulin gene was hybridized to human chromosome preparations. A high proportion (25%) of hybridized metaphases exhibited silver grains at the distal portion of the long arm of chromosome 8. Analysis of the grain position at this site indicated that the chromosomal localization of the human thyroglobulin gene was 8q242-8q243.     

Identification of a human heart FABP pseudogene located on chromosome 13

The fatty acid-binding proteins (FABPs) constitute a conserved group of cytosolic low molecular mass proteins, which consists of several types: liver, heart, myelin, epidermal, adipocyte, brain, intestinal and ileal type. The FABP gene structure is well conserved during evolution and exhibits a four-exon/three-intron structure. In the past, multiple hybridizing fragments were detected upon Southern blot analysis using heart FABP (H-FABP) cDNA as a probe. The origin of these fragments was not clear. We screened a human genomic library and isolated an intronless gene (FABP3-ps) with 85% similarity to the human H-FABP cDNA and high similarity (76 and 79%) to the H-FABP cDNAs of mouse and bovine, respectively. By means of fluorescence in situ hybridization this processed pseudogene could be assigned chromosome 13q13-q14, whereas the gene for human H-FABP (FABP3) resides on chromosome 1p32-p33. No expression of the processed pseudogene could be detected in skeletal muscle or fetal brain.     

Molecular cloning, mapping and characterization of the human neurocalcin delta gene (NCALD)

We identified a new human gene that encodes a cognate of the bovine neurocalcin delta from a human fetal brain cDNA library; hence we named it human neurocalcin delta (NCALD) gene. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 100% and 99% identical to that of the bovine and chicken neurocalcin, respectively. Northern blots showed that the NCALD gene is more abundantly expressed in brain, testis, ovary and small intestine. Tissue in situ hybridization confirmed the existence of the NCALD mRNA in the adult human testis. Radiation hybrid panel mapping localized the gene to chromosome 8 between molecular markers D8S270 and D8S257.     

The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas

A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.     

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.     

Chromosomal localization of the gene for human glucosamine-6-sulphatase to 12q14

Summary Glucosamine-6-sulphatase (G6S), a lysosomal enzyme found in all cells, is involved in the catabolism of heparin, heparan sulphate, and keratan sulphate. Deficiency of G6S results in the accumulation of undegraded substrate and the lysosomal storage disorder mucopolysaccharidosis type IIID (Sanfilippo D syndrome). Regional mapping by in situ hybridization of a ³H-labelled human G6S cDNA probe to human metaphase chromosomes indicated that the G6S gene is localized to chromosome 12 at q14. The localization of the G6S gene to chromosome 12 was confirmed using the G6S cDNA clone in Southern blot hybridization analysis of DNA from human x mouse hybrid cell lines.