Cloning and Gene Mapping of the Mouse Homologue of theCBFA2T1Gene Associated with Human Acute Myeloid Leukemia

The humanCBFA2T1(also known asMTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of theCBFA2T1gene,Cbfa2t1h.Nucleotide sequence analysis ofCbfa2t1hcDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5′- and 3′-untranslated regions (87.0, 92.0, and 93.7% identities for 5′-untranslated, coding, 3′-untranslated regions, respectively). The 3′-untranslated region ofCbfa2t1hcontains a (CA)_ndinucleotide repeat, and the polymerase chain reaction amplification of the (CA)_nrepeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mappedCbfa2t1hto mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 × Mae) × Mae cross. The chromosomal location was also confirmed by fluorescencein situhybridization.     

Characterization of Three Novel Human Cadherin Genes (CDH7, CDH19, and CDH20) Clustered on Chromosome 18q22–q23 and with High Homology to Chicken Cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5′ and 3′ rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3′ RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse “cadherin-7” cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22–q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

Identification of Neurofibromatosis 1 (NF1) Homologous Loci by Direct Sequencing, Fluorescence in Situ Hybridization, and PCR Amplification of Somatic Cell Hybrids

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1 -homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5′ end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template.     

Chemokine PARC Gene (SCYA18) Generated by Fusion of Two MIP-1α/LD78α-like Genes

Two loci in the human genome, chromosomes 4q12–q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1α (MIP-1α)/LD78α, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1α. Analyses of the BAC clones containing the humanPARCgene indicated that the gene is located most closely toMIP-1α(HGMW-approved symbolSCYA3) andMIP-1β(HGMW-approved symbolSCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that thePARCgene had been generated by fusion of twoMIP-1α-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of theMIP-1αgene calledLD78β(HGMW-approved symbolSCYA3L) in the vicinity of theMIP-1αgene, the locus surrounding theMIP-1αgene seems to be a “hot spring” that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.     

Comparative Genome Mapping of the Ataxia–Telangiectasia Region in Mouse, Rat, and Syrian Hamster

Chromosomal locations of theAtm(ataxia–telangiectasia (AT)-mutated) andAcat1(mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4–qa5 of Syrian hamster chromosome 12. The regions in the mouse and rat were homologous to human chromosome 11q. Fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice.Atm, Acat1,andNpat,which is a new gene isolated from the AT region, and 12 flanking microsatellite DNA markers were examined. No recombinations were found among theAtm, Npat, Acat1,andD9Mit6loci, and these loci were mapped 2.0 cM distal toD9Mit99and 1.3 cM proximal toD9Mit102.Comparison of the linkage map of mouse chromosome 9 (MMU9) and that of human chromosome 11 (HSA11) indicates that there is a chromosomal rearrangement due to an inversion betweenEts1andAtm–Npat–Acat1and that the inversion of MMU9 originated from the chromosomal breakage at the boundary betweenGria4andAtm–Npat–Acat1on HSA11. This type of inversion appeared to be conserved in the three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with theRckgene.     

Recombinational and Physical Mapping of the Locus for Primary Open-Angle Glaucoma (GLC1A) on Chromosome 1q23–q25

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness in industrialized countries. A locus for juvenile-onset POAG,GLC1A,has been mapped to 1q21–q31 in a 9-cM interval. With recombinant haplotypes, we have now reduced theGLC1Ainterval to a maximum of 3 cM, between theD1S452/NGA1/D1S210andNGA5loci. These loci are 2.8 Mb apart on a 4.7-Mb contig that we have completed between theD1S2851andD1S218loci and that includes 96 YAC clones and 48 STSs. The newGLC1Ainterval itself is now covered by 25 YACs, 30 STSs, and 16 restriction enzyme site landmarks. The lack of aNotI site suggests that the region has few CpG islands and a low gene content. This is compatible with its predominant cytogenetic location on the 1q24 G-band. Finally, we have excluded important candidate genes, including genes coding for three ATPases (ATP1B1, ATP2B4, ATP1A2), an ion channel (VDAC4), antithrombine III (AT3), and prostaglandin synthase (PTGS2). Our results provide a basis to identify theGLC1Agene.     

Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation). NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion. To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome. In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22. We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene. However, the GAP-related domain itself is not represented in this locus. In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21. These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus. Received: 18 December 1995 / Revised: 5 February 1996     

The leukemia associated <Emphasis Type="Italic">ETO</Emphasis> nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

Background  

The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.     

Localization ofHuC(ELAVL3) to Chromosome 19p13.2 by Fluorescencein SituHybridization Utilizing a Novel Tyramide Labeling Technique

HuC is a neural-specific member of the Elav family of RNA-binding proteins. This highly conserved gene family plays a crucial role in neurogenesis, andHuC(HGMW-approved symbol ELAVL3) is expressed at an early stage of neural development. Using a novel tyramide fluorescencein situhybridization (T-FISH) technique, we localizedHuCto chromosome 19p13.2. This localization was confirmed by radiation hybrid mapping and coincides with that ofHuR(HGMW-approved symbol ELAVL1), anotherelavfamily member. Dual T-FISH analysis withHuCandHuRprobes, however, indicated distinct loci, withHuCbeing centromeric to HuR. This study demonstrates the utility of T-FISH in colocalizing two genes on the same chromosomal preparation using only biotinylated probes.     

Human Ovalbumin Serpin Evolution: Phylogenic Analysis, Gene Organization, and Identification of New PI8-Related Genes Suggest That Two Interchromosomal and Several Intrachromosomal Duplications Generated the Gene Clusters at 18q21–q23 and 6p25

The human ovalbumin (ov) serpins are associated with tumorigenesis, inflammation, and protection from autolysis by granule proteinases. Their genes are located at 18q21 or 6p25, falling into two structurally very similar but distinct categories depending on the presence or absence of a particular exon. Analysis of ov-serpin gene structure provides an opportunity to elucidate the mechanisms contributing to the formation of the larger serpin gene superfamily. Here we have identified a new gene (PI8L1) at 6p25 that is 72% identical to the 18q21 gene PI8. FISH analysis using the 3′ untranslated region of PI8 yielded an additional signal at 18q23, separable from the known 18q21.3 signal by the t(1;18)(p32;q23) chromosomal translocation. The presence of more than one PI8-related gene was confirmed by analysis of human genomic DNA using the same probe. Cloning and analysis of PI8 showed that its intron number and phasing are identical to those of the 6p25 genes PI6, PI9, and ELANH2, and it lacks the interhelical variable loop exon found in other 18q21 genes. PCR analysis demonstrated that PI5 at 18q21 also lacks this exon, indicating that it is organized identically to the 6p25 genes. By contrast, PI10 and megsin have this exon and resemble the other 18q21 genes, PLANH2, SCCA-1, and SCCA-2, in structure. Using these data with an ov-serpin phylogenic tree we have constructed, we propose that the ov-serpin gene clusters arose via interchromosomal duplication of PI5 (or a precursor) to 6p25, followed by duplication at 6p25, and a more recent interchromosomal duplication from 6p25 to 18q to yield PI8.     

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.