Physical and transcriptional map of a 311-kb segment of chromosome 18q21, a candidate lung tumor suppressor locus.

Here, we report the complete genomic sequence and the characterization of the 311-kb region of 18q21, a candidate tumor suppressor locus containing a region of homozygous deletion in a lung cancer cell line, Ma29. This region contained two known genes, SMAD4 and ME2 (mitochondrial malate oxydoreductase), and two novel genes, D29 (deleted in Ma29 HGMW-approved symbol ELAC1), encoding an evolutionarily conserved protein, and B29 (beside the Ma29 deletion HGMW-approved symbol C18orf3), with no significant homology to any known genes. The deleted DNA segment in Ma29, which was estimated to be 195 kb in size, included all the coding exons of ME2 and D29, but not the coding exons of SMAD4 and B29. The deleted region also included exon 0, a 5'-noncoding exon, of SMAD4, and the expression of SMAD4 was greatly reduced in Ma29 cells. Mutations of SMAD4 and D29 were detected in 1 of 45 lung cancer cell lines examined, while those of ME2 and B29 were not detected, indicating that these four genes are not major targets for 18q21 deletions. The physical and transcriptional map constructed in this study will provide basic information for the identification of a tumor suppressor gene(s) at 18q21 involved in lung carcinogenesis.

Genomic anatomy of a premier major histocompatibility complex paralogous region on chromosome 1q21-q22.

Human chromosomes 1q21-q25, 6p21.3-22.2, 9q33-q34, and 19p13.1-p13.4 carry clusters of paralogous loci, to date best defined by the flagship 6p MHC region. They have presumably been created by two rounds of large-scale genomic duplications around the time of vertebrate emergence. Phylogenetically, the 1q21-25 region seems most closely related to the 6p21.3 MHC region, as it is only the MHC paralogous region that includes bona fide MHC class I genes, the CD1 and MR1 loci. Here, to clarify the genomic structure of this model MHC paralogous region as well as to gain insight into the evolutionary dynamics of the entire quadriplication process, a detailed analysis of a critical 1.7 megabase (Mb) region was performed. To this end, a composite, deep, YAC, BAC, and PAC contig encompassing all five CD1 genes and linking the centromeric +P5 locus to the telomeric KRTC7 locus was constructed. Within this contig a 1.1-Mb BAC and PAC core segment joining CD1D to FCER1A was fully sequenced and thoroughly analyzed. This led to the mapping of a total of 41 genes (12 expressed genes, 12 possibly expressed genes, and 17 pseudogenes), among which 31 were novel. The latter include 20 olfactory receptor (OR) genes, 9 of which are potentially expressed. Importantly, CD1, SPTA1, OR, and FCERIA belong to multigene families, which have paralogues in the other three regions. Furthermore, it is noteworthy that 12 of the 13 expressed genes in the 1q21-q22 region around the CD1 loci are immunologically relevant. In addition to CD1A-E, these include SPTA1, MNDA, IFI-16, AIM2, BL1A, FY and FCERIA. This functional convergence of structurally unrelated genes is reminiscent of the 6p MHC region, and perhaps represents the emergence of yet another antigen presentation gene cluster, in this case dedicated to lipid/glycolipid antigens rather than antigen-derived peptides.

Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region.

The intensely studied MHC has become the paradigm for understanding the architectural evolution of vertebrate multigene families. The 4-Mb human MHC (also known as the HLA complex) encodes genes critically involved in the immune response, graft rejection, and disease susceptibility. Here we report the continuous 1,796,938-bp genomic sequence of the HLA class I region, linking genes between MICB and HLA-F. A total of 127 genes or potentially coding sequences were recognized within the analyzed sequence, establishing a high gene density of one per every 14.1 kb. The identification of 758 microsatellite provides tools for high-resolution mapping of HLA class I-associated disease genes. Most importantly, we establish that the repeated duplication and subsequent diversification of a minimal building block, MIC-HCGIX-3.8-1-P5-HCGIV-HLA class I-HCGII, engendered the present-day MHC. That the currently nonessential HLA-F and MICE genes have acted as progenitors to today's immune-competent HLA-ABC and MICA/B genes provides experimental evidence for evolution by "birth and death," which has general relevance to our understanding of the evolutionary forces driving vertebrate multigene families.

An analysis of 114 patients with dermal melanocytosis on the trunk and extremities.

Dermal melanocytosis is commonly treated with Q-switched neodymium:yttrium-aluminium-garnet (Nd:YAG) laser today. We report on the clinical effect of laser treatment of dermal melanocytosis of the trunk and extremities in 114 patients. The effect of numbers of such treatment was assessed by clinical examination and classified into six grades. All the patients who had more than eight treatments achieved more than 75% clearance. Deep-blue with brown speckled lesions tended to clear more easily than uniform deep-blue lesions. We expect that all dermal melanocytosis of the trunk and extremities can be cleared after a maximum of 12 treatments.

Mapping of a novel human carbonyl reductase, CBR3, and ribosomal pseudogenes to human chromosome 21q22.2.

To find the genes contributing to Down syndrome, we constructed a 4-Mb sequence-ready map spanning chromosome 21q22.2 with megabase-sized cosmid/P1-derived artificial chromosome (PAC) contigs. The restriction map with rare cutting enzymes, followed by sequencing from the clustering sites, has defined CpG islands and revealed the genes associated with CpG islands (Accession No. D85771). Of these, two human carbonyl reductases (CBR; EC1.1.1.184) were found in a PAC 25P16 clone. CBR catalyzes the reduction of a large number of biologically and pharmacologically active carbonyl compounds to their corresponding alcohols and has been mapped in 21q22.1. To confirm these results, we sequenced the PAC clone in shotgun strategies and identified a novel carbonyl reductase, designated CBR3, 62 kb downstream from the original CBR. In addition, three ribosomal pseudogenes, L23a, S9, and L3, and some cDNAs with ESTs were mapped in the sequence. In conclusion, the sequence analysis for CpG islands predicted from the megabase-sized contigs will reveal and identify the genes involved in Down syndrome.

Nucleotide sequencing analysis of the 146-kilobase segment around the IkBL and MICA genes at the centromeric end of the HLA class I region.

To elucidate the complete gene structure and to identify new genes involved in the development of HLA class I antigen-associated diseases in the class I region of the human major histocompatibility complex on chromosome 6, a YAC clone (745D12) covering the 146-kb segment around the IkBL and MICA loci was isolated from a YAC library constructed from the B-cell line, BOLETH. A physical map of this region was constructed by isolation of overlapping cosmid clones derived from 745D12. Of these, five contiguous cosmids were chosen for DNA sequencing by the shotgun strategy to give a single contig of 146,601 bp from 2.8 kb telomeric of the IkBL gene to exon 6 of MICA. This region was confirmed to contain five known genes, IkBL, BAT1, MICB, P5-1, and HLA-X (class I fragment), from centromere to telomere, and their exon-intron organizations were determined. The 3.8-1 homologue gene (3.8-1-hom) showing 99.7% identity with the 3.8-1 cDNA clone, which was originally isolated using the 3.8-kb EcoRI fragment between the HLA-54/H and the HLA-G genes, was detected between MICA and MICB and was suggested to represent the cognate 3.8-1 genomic sequence from which the cDNA clone was derived. No evidence for the presence of expressed new genes could be obtained in this region by homology and EST searches or coding and exon prediction analyses. One TA microsatellite repeat spanning 2545 bases with as many as 913 repetitions was found on the centromeric side of the MICA gene and was indicated to be a potential hot spot for genetic recombination. The two segments of approximately 35 kb upstream of the MICA and MICB genes showed high sequence homology (about 85%) to each other, suggesting that segmental genome duplication including the MICA and MICB genes must have occurred during the evolution of the human MHC.

Nucleotide sequence analysis of the HLA class I region spanning the 237-kb segment around the HLA-B and -C genes.

To elucidate the detailed gene organization of the human leukocyte antigen (HLA) class I region on chromosome 6, seven contiguous cosmid genomic clones covering the 237-kb segment around the HLA-B and -C loci were subjected to DNA sequencing by the shotgun strategy to give a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C. This region was confirmed to contain four known genes, MICA, HLA-17, HLA-B, and HLA-C, from centromere to telomere. Further, a new member of the P5 multicopy genes was found to be about 1.3 kb upstream of the HLA-17 gene and designated P5.8. Five novel genes designated NOB1-5 were identified by RT-PCR and Northern blot hybridization. In addition, two pseudogenes, dihydrofolate reductase pseudogene (DHFRP) and ribosomal protein L3 homologous gene (RPL3-Hom), were also found in the vicinity of the HLA-B and -C genes, respectively. The two segments (about 40 kb) downstream of the HLA-B and HLA-C genes showed high sequence homology to each other, suggesting that segmental genome duplication including the major histocompatibility complex (MHC) class I gene must have occurred during the evolution of the MHC.

Antinociceptive effect of L-arginine on the carrageenin-induced hyperalgesia of the rat: possible involvement of central opioidergic systems.

L-arginine is considered to be a precursor substance of kyotorphin (tyrosyl-arginine), a [Met5]enkephalin releaser with antinociceptive action. We examined the antinociceptive effect of L-arginine in rats. L-Arginine (300-1000 mg/kg) administered subcutaneously (s.c.) elicited antinociception (assessed by the Randall-Selitto method) in rats with a carrageenin-treated hindpaw. Naloxone (2 mg/kg s.c.) but not N-methyl-levallorphan (20 mg/kg s.c.), a peripherally selective opioid antagonist, inhibited L-arginine-induced antinociception. Intracerebroventricular administration of L-arginine (0.2-1.0 mg/rat) produced a dose-related inhibition of the carrageenin-induced hyperalgesia. Intraplantar (i.pl.) injection of L-arginine (0.5-1.0 mg/paw) also induced antinociception, which was resistant to naloxone (2 mg/kg s.c.) but was antagonized by methylene blue (0.5 mg/paw i.pl.), a guanylate cyclase inhibitor. L-Arginine (1000 mg/kg s.c.) did not inhibit edema formation in the carrageenin-treated rat hindpaw. These results suggest that systemically administered L-arginine produces mainly an antinociceptive effect mediated by central opioidergic mechanisms in rats with carrageenin-induced hyperalgesia.