The genomic organization of type I keratin genes in mice

We isolated two new keratin cDNAs by screening a cDNA library constructed from poly(A)+ RNA of the dorsal and abdominal skin of C57BL/10J mice with a probe of human KRT14. Due to its high sequence homology to human keratin 17 cDNA, one full-length cDNA is most likely to be mouse keratin 17 (Krt1-17) cDNA. The other is the putative full-length cDNA of a novel type I keratin gene, designated Krt1-c29. These two keratin genes were mapped to the distal portion of Chromosome 11, where the mouse keratin gene complex-1 (Krt1) is localized. To elucidate the genomic organization of Krt1 in mice, we carried out genetic and physical analyses of Krt1. A large-scale linkage analysis using intersubspecific backcrosses suggested that there are two major clusters in Krt1, one containing Krt1-c29, Krt1-10, and Krt1-12 and the other containing Krt1-14, -15, -17, and -19. Truncation experiments with two yeast artificial chromosome clones containing the two clusters above have revealed that the gene order of Krt1 is centromere-Krt1-c29-Krt1-10-Krt1-12-Krt1-13-K rt1-15-Krt1-19-Krt1-14-K rt1-17-telomere. Finally, we analyzed sequence divergence between the genes belonging to the Krt1 complex. The results clearly indicated that genes are classified into two major groups with respect to phylogenetic relationship. Each group consists of the respective gene cluster demonstrated by genetic and physical analyses in this study, suggesting that the physical organization of the Krt1 complex reflects the evolutionary process of gene duplication of this complex.     

The structure-activity relationship study on 2-, 5-, and 6-position of the water soluble 1,4-dihydropyridine derivatives blocking N-type calcium channels

In order to find an injectable and selective N-type calcium channel blocker, we have performed the structure–activity relationship (SAR) study on the 2-, 5-, and 6-position of 1,4-dihydropyridine-3-carboxylate derivative APJ2708 (2), which is a derivative of Cilnidipine and has L/N-type calcium channel dual inhibitory activities. As a consequence of the optimization, 6-dimethylacetal derivative 7 was found to have an effective inhibitory activity against N-type calcium channels with more than 170-fold lower activity for L-type channel compared to that of APJ2708.     

Fine mapping and allelic dosage effect of <Emphasis Type="Italic">Hwc1</Emphasis>, a complementary hybrid weakness gene in rice

Hybrid weakness is a reproductive barrier that is found in many plant species. In rice, the hybrid weakness caused by two complementary genes, Hwc1 and Hwc2, has been surveyed intensively. However, their gene products and the molecular mechanism that causes hybrid weakness have remained unknown. We performed linkage analyses of Hwc1, narrowed down the area of interest to 60 kb, and identified eight candidate genes. In the F(2) population, in which both Hwc1 and Hwc2 genes were segregated, plants were separable into four classes according to their respective phenotypes: severe type, semi-severe type, F(1) type, and normal type. Severe type plants show such severe symptoms that they could produce only tiny shoot-like structures; they were unable to generate roots. Genetic analyses using closely linked DNA markers of the two genes showed that the symptoms of the F(2) plants were explainable by the genotypes of Hwc1 and Hwc2. Weakness was observed in plants that have both Hwc1 and Hwc2. In Hwc1 homozygote, the symptoms worsened and severe type or semi-severe type plants appeared. Consequently, Hwc1 should have a gene dosage effect and be a semi-dominant gene. The dosage effect of Hwc2 was recognizable, but it was not so severe as that in Hwc1. These results are useful to elucidate the mechanism that causes the hybrid weakness phenomenon and the role of each causal gene in hybrid weakness.     

Microsatellite variation in Japanese and Asian horses and their phylogenetic relationship using a European horse outgroup

The genetic relationships of seven Japanese and four mainland-Asian horse populations, as well as two European horse populations, were estimated using data for 20 microsatellite loci. Mongolian horses showed the highest average heterozygosities (0.75-0.77) in all populations. Phylogenetic analysis showed the existence of three distinct clusters supported by high bootstrap values: the European cluster (Anglo-Arab and thoroughbreds), the Hokkaido-Kiso cluster, and the Mongolian cluster. The relationships of these clusters were consistent with their geographical distributions. Basing our assumptions on the phylogenetic tree and the genetic variation of horse populations, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The genetic relationship of Japanese horses corresponded to their geographical distribution. Microsatellite polymorphism data were shown to be useful for estimating the genetic relationships between Japanese horses and Asian horses.     

Evolutionary genomics: molecular evolution at the genomic scale

The Symposium on Evolutionary Genomics was held in Atami, Japan, from 4 to 6 November 2001.     

Inhibition of the human chemokine receptor CXCR4 by antisense phosphorothioate oligodeoxyribonucleotides

The CXC chemokine receptor CXCR4/fusion, a major coreceptor for the T-cell line T-tropic (X4) HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. In the present study, we describe the effects of an antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN) on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for human immunodeficiency virus type 1 (HIV-1) infection. The naked antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN-1), containing the AUG initiation codon at the center of the oligodeoxyribonucleotide, showed a slightly higher inhibitory effect on HIV-1 gag p24 production among all sequences tested. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for the delivery of the anti-S-ODN-1. The anti-S-ODN-1 encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN-1. When the anti-S-ODN-1 encapsulated with RNP was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor showed high suppression, indicating sequence-specific inhibition. The activities of unmodified oligodeoxyribonucleotide are effectively enhanced by using a basic peptide, RNP.     

Mitochondrial DNA polymorphism among five Asian populations

Mitochondrial DNA (mtDNA) polymorphisms were detected using 13 restriction enzymes on the total DNA obtained from blood samples of five Asian populations: Japanese and Ainu of northern Japan, Korean, Negrito (Aeta) of the Philippines, and Vedda of Sri Lanka. Of a total of 28 restriction-enzyme morphs detected, eight had not been reported previously. By combining the morphs, we were able to classify mtDNAs of 243 individuals into 20 mtDNA types. Phylogenetic analyses using maximum parsimony and genetic distance methods both showed that the Japanese, Ainu, and Korean populations were closely related to each other. Aeta was found to show a relatively close relationship to these three populations, confirming the conclusion from previous studies of blood markers. In contrast, Vedda was quite different from the other four populations.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.