J. Inazawa

High resolution ordering of DNA markers by multi-color fluorescent in situ hybridization of prophase chromosomes

To improve resolution for physical ordering of adjacent DNA loci, prophase chromosomes were used for multi-color fluorescent in situ hybridization (FISH). The prophase chromosomes were prepared from cultured lymphocytes by a thymidine synchronization, bromodeoxyuridine release technique and then treating the synchronized cultures with topoisomerase II inhibitors ICRF154 or ICRF193. Almost all mitotic figures exhibited highly elongated prophase chromosomes without significant reduction of the mitotic index. Using multi-color FISH with these prophase chromosomes, we were able to distinguish signals for loci separated by as little as 50 kb, and determine their orientation. Furthermore, using this prophase ordering system, we confirmed the linear order and defined the orientation of seven cosmid markers within a 360-kb region surrounding D10S102, a locus that is closely linked to the disease locus in families segregating an allele causing multiple endocrine neoplasia IIA (MEN2A). This prophase FISH system, by rapidly and precisely providing the linear order of loci that are very close, can expedite construction of fine cytogenetic maps and contribute to positional-cloning studies in which the precise ordering of DNA loci in a target region is critical.

The human homologue of the murine Llglh gene (LLGL) maps within the Smith-Magenis syndrome region in 17p11.2.

We have isolated and characterized the human homologue of the murine Llglh gene, which was originally isolated as a homologue of a Drosophila tumor suppressor gene 1(2)gl (lethal(2) giant larvae). In the mouse, Llglh is thought to play an important role during brain development as a regulatory target of Hoxc8. The human homologue of Llglh (LLGL) encodes a protein consisting of 1,033 amino acids. This gene was mapped by fluorescence in situ hybridization (FISH) to human chromosome 17p11.2, a region that is typically deleted in patients with Smith-Magenis syndrome (SMS). In our FISH analysis of metaphase chromosomes of four SMS patients, a probe representing LLGL failed in each case to hybridize to one of the two chromosome 17 homologues, indicating that this gene may play a role in the pathogenesis of SMS.

Molecular cloning and mapping of a human cDNA (SC5DL) encoding a protein homologous to fungal sterol-C5-desaturase

We have isolated a human cDNA clone homologous to fungal ERG3, a gene encoding sterol C-5 desaturase. The full nucleotide sequence of this human cDNA revealed a 708-bp open reading frame that encodes a 236-amino-acid polypeptide. The gene was expressed in all normal human tissues examined. We determined its location to chromosome 11q23.3 by fluorescence in situ hybridization.

Molecular cloning of CISH, chromosome assignment to 3p21.3, and analysis of expression in fetal and adult tissues

The murine cytokine inducible SH2-containing (Cis) protein gene (Cish) was recently cloned and shown to have a growth inhibitory function. We have isolated cDNAs coding for its human homologue (CISH) and assigned the gene to 3p21.3 by fluorescence in situ hybridization. Northern blot analysis showed CISH expression in various epithelial tissues including lung and kidney, in which tumors frequently exhibit 3p21.3 deletions.

Isolation and mapping of a human gene (RPD3L1) that is homologous to RPD3, a transcription factor in Saccharomyces cerevisiae

We have isolated a novel human gene RPD3L1, that is highly homologous to a transcription factor in Saccharomyces cerevisiae, RPD3 (reduced potassium dependency 3), from a human fetal lung cDNA library. The cDNA clone, hRPD3, consists of 2,100 nucleotides that contain an open reading frame of 1446 nucleotides encoding 482 amino acids. It shares 62% identity in nucleotide sequence and 52% identity in amino acid sequence to RPD3. This gene is expressed at various levels in all tissues examined. Furthermore, we were able to map it to chromosome band 1p34.1 by FISH.

Assignment of the human myeloperoxidase gene (MPO) to bands q21.3→q23 of chromosome 17

Using a human myeloperoxidase cDNA, we have mapped the human myeloperoxidase gene to chromosome 17 at q21.3----q23 by in situ hybridization to metaphase chromosomes from human lymphocyte preparations.

Assignment of the human renal dipeptidase gene (DPEP1) to band q24 of chromosome 16

Renal dipeptidase (DPEP1) or dehydropeptidase-I (E.C.3.4.13.11) is a kidney membrane enzyme which hydrolyzes a variety of dipeptides. DPEP1 is implicated in the renal metabolism of glutathione and its conjugates and is also responsible for hydrolysis of beta-lactam antibiotics. Using a human DPEP1 cDNA probe, we mapped the human renal dipeptidase gene to human chromosome 16 at band q24 by in situ hybridization.

Precise ordering of 26 cosmid markers on chromosome region 3p23→p21.3 by two-color FISH on human prophase chromosomes and stretched DNAs

To construct a detailed cytogenetic map of human chromosome region 3p23-->p21.3, we determined the order of 26 cosmid markers (cCI 3 series) previously localized within this region by fluorescence in situ hybridization (FISH). Two-color pairwise FISH analysis of prophase chromosomes provided the order of these markers as follows: pter - 245 (D3S647) - 872 (D3S1018) - 818 (D3S996) - 905 (D3S1022) - 515 (D3S685) - 1195 (D3S1125) - 718 (D3S935) - 911 (D3S1025) - 878 (D3S1020) - 717 (D3S934) - 401 (D3S664) - [708 (D3S926)/524 (D3S686)] - 848 (D3S1011) - 771 (D3S966) - 917 (D3S1029) - 533 (D3S688) - 470 (D3S676) - 940 (D3S1037) - 785 (D3S974) - 810 (D3S988) - 9 (D3S643) - 382 (D3S660) - 769 (D3S965) - 792 (D3S978) - 604 (D3S705) - cen. The two-color signals of 524 (D3S686) and 708 (D3S926) were visualized as an overlapping pattern on prophase chromosomes, and, further, the string signals also overlapped on stretched DNAs, allowing us to determine their precise order as pter - D3S926 - D3S686 - cen. The precise order of 26 DNA markers on 3p23-->p21.3 can provide useful information for the positional cloning of tumor suppressor gene(s) and cancer breakpoint(s) encompassed in this region.

Isolation and mapping of a human gene (RABL) encoding a small GTP-binding protein homologous to the Ras-related RAB gene.

Many subgroups of the RAB gene, a member of the RAS superfamily have been identified. Here we report the isolation and analysis of a cDNA encoding a putative small GTP-binding protein, designated RABL, from a human fetal lung cDNA library. RABL encodes 216 amino acids that are 86% identical to members of the RAB5 subfamily and it shows 94% homology in nucleotide sequence with RAB5C of dog. This gene was expressed ubiquitously in all human tissues examined. By fluorescence in situ hybridization we mapped RABL to chromosome band 17q21.2.

Molecular cloning of a human cDNA encoding putative cysteine protease (PRSC1) and its chromosome assignment to 14q32.1

We have isolated a novel human cDNA encoding a protein of 433 amino acids which shows 40% sequence identity to a hemoglobinase of Schistosoma japonicum, one of the cysteine proteases in the pathway by which trematodes degrade host-cell globin. It also has 36% identity to a cysteine protease of the soybean Glycine max that processes proproteins of various vacuolar proteins into mature form after translation. Considering the sequence similarities among these proteins in such widely divergent species, the human cDNA reported here appears to represent one of a group of genes that have been well conserved during evolution. Northern-blot analysis revealed the strongest expression of this gene in kidney, among the human tissues examined. By fluorescence in situ hybridization, we determined its chromosome location at 14q32.1.