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PubMed1994

Structural organization of the human tyrosinase gene and sequence analysis and characterization of its promoter region.

Tyrosinase is the principal enzyme in the biosynthesis of melanin. The expression of tyrosinase is tissue-specific and appears to be regulated by various hormonal and environmental factors. Elucidation of the genomic structure and molecular basis of control of tyrosinase gene expression will greatly enhance our understanding of the regulation of human pigmentation. To this end, we have isolated and performed restriction mapping of recombinant cosmid and lambda phage clones containing the human tyrosinase gene, sequenced a 2.2-kilobase (kb) region of its promoter, and determined the potential regions regulating the tyrosinase gene expression in transient-expression system. The human tyrosinase gene is comprised of five exons and four introns. Based on our restriction mapping studies, the gene spans a distance of over 65-kb on chromosome 11 (q14-->q21). We constructed a series of plasmids (pHTY-CAT) that contain 5' sequential deletions of the human tyrosinase 5' flanking sequence fused to the reporter gene, chloramphenicol acetyltransferase (CAT). The plasmids were used to locate promoter regions that are potential regulators of tyrosinase gene expression in a transient expression system using melanoma cell lines. In human melanoma cells, the plasmid construct with a -2020 base pair (bp) promoter yielded the highest CAT activity. When the deletions reached -1739 bp, the CAT activity was dramatically reduced, indicating that important enhancer elements for transcription control are present between -1739 and -2020 bp. Further deletions up to -550 bp also resulted in dramatic decreases of CAT activity. However, when the deletion included -550 bp of the 5' flanking sequence, there was 26 percent of the CAT activity compared to that of the -2020 bp promoter. Deletions beyond -550 bp also showed markedly decreased CAT activity. Based on our data, we suggest that human tyrosinase gene expression is governed by both tissue-specific and multiple regulatory elements.

Base Sequence↗

PubMed1998

DNA polymorphisms at alpha-Gpdh locus of Drosophila melanogaster in Korean population.

Restriction site variation in 18-kb region including the Gpdh (sn-glycerol-3-phosphate dehydrogenase) locus has been assessed in 27 isofemale lines, 12 lines from the Chounan population and 15 lines from the Pusan population in Korea. Of the 25 restriction sites that were scored, 5 sites were polymorphic (20.0%): PstI (-2.35), XbaI (-1.7), HindIII (0.0), HindIII (+0.25), and PstI (+5.7). Two insertions (I) and three deletions (D) were detected. All the restriction sites and length polymorphisms were located in introns and outside regions of the Gpdh structure gene, the area from -7.0 site to -5.5 site. The proportion of segregating nucleotides, p, for all 27 lines was 0.019 and the estimated heterozygosity per nucleotide pair, was 0.013. Sixteen kinds of restriction map haplotypes of all lines analyzed were detected. Few lines were identical at the restriction map level. The estimated haplotype diversity, h, was 0.968. The haplotype diversity (0.997) of GpdhFF bearing chromosomes was slightly higher than that (0.982) of GpdhSS chromosomes. Analysis for a linkage disequilibria between the polymorphism also showed that HindIII (+0.25) vs. I (a) and PstI (-2.35) vs. D (a) have relatively high level of the linkage disequilibria. There were no significant relationship between restriction site polymorphism and GPDH activity in 27 second-chromosome lines; however, there was an apparent difference in the effect of length variation on GDPH activity. The activity of deletion-bearing lines was lower than that of deletion-free lines, while there was no effect of insertion on activity. Duplication of Gpdh was observed in about one-third of the 27 isofemale lines. The duplication-bearing lines in both populations had higher levels of GPDH activity and GPDH CRM than duplication-free lines. In a further investigation of the duplication structure, the duplication of Korean populations was shown to have a similar structure to other populations investigated (Symonds and Gibson, 1992; Takano et al., 1989).

Animals↗

PubMed1993

The origin and genetic differentiation of native breeds of pigs in southwest China: an approach from mitochondrial DNA polymorphism.

Mitochondrial DNA (mtDNA) of six breeds of native domestic pigs from Yunnan province, southwest China, and two wild boars obtained from Sichuan, China, and Vietnam was analyzed using 20 restriction endonucleases that recognize six nucleotides. Restriction maps were made by double-digestion methods and polymorphic sites were located on the map. According to their mtDNA restriction types, all the breeds were classified into six groups. Genetic distances among groups were calculated to define their phylogenetic relationships. The relationship between the Sichuan wild boar and domestic pigs is close, while the Vietnamese wild boar is relatively far from them, so the domestic pigs in southwest China are likely to have originated from a wild pig which distributed in west China. We compare our results with previous reports in literature and discuss the relationship among Chinese pigs, Japanese pigs, and European pigs. The mtDNA cleavage pattern of the Mingguang pig digested by EcoRV was identical to that of Duroc; mutations at the EcoRI site, detected in the mtDNA of two Dahe pigs, are the same as in the Vietnamese wild boar, suggesting that mutational hot spots exist in the mtDNA of pigs.

Animals↗

PubMed1992

Five major nuclear ribosomal repeats represent a large and variable fraction of the genomic DNA of Picea rubens and P. mariana.

The nuclear ribosomal repeats for the 18S, 5.8S, and 26S RNAs of two closely related Picea (spruce) species were characterized by restriction mapping and Southern blot hybridization. Restriction polymorphisms were identified in the IGS and ITS sequences; however, no polymorphism was species specific. As many as five different rDNA repeat units were observed in individual genomes. The repeat size for these gymnosperms ranged from a minimum of 32 kbp to greater than 40 kbp, two- to threefold larger than the typical angiosperm rDNA unit. Slot-blot hybridizations were used to determine the nuclear rDNA copy concentration. Among P. rubens individuals threefold variation was observed in the rDNA copy concentration, and among P. mariana individuals such variation was as much as sixfold. At a size greater than 32 kbp and at a concentration averaging 1.2-1.3 x 10(4) copies/pg, the rDNA constitutes approximately 4% of the total genome. Regression analysis revealed a significant relationship between copy concentration of the rDNA repeat unit in P. rubens and geographic origins. Differences in the rDNA content in Picea could contribute to the variation, in overall genome size, that has been observed within conifer species.

Base Sequence↗

PubMed1992

Characterization of a plasmid from moderately halophilic eubacteria.

A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.

Drug Resistance, Microbial↗

PubMed1981

Cloning of a recA-like gene of Proteus mirabilis.

A gene of Proteus mirabilis that can substitute for functions of the recA gene of Escherichia coli has been cloned into the plasmid pBR322, using shotgun experiments. The recA-like gene (recAp.m.) has been localized by restriction mapping within a 1.5-Md PstI fragment that is a part of two cloned HindIII fragments of the chromosome of P. mirabilis. The restriction map of the recAp.m. gene differs from that of the recA gene of E. coli. Functionally, the recombinant plasmids containing the recAp.m. gene restore a nearly wild-type level of UV-resistance to several point and deletion mutants in the recA gene of E. coli.

Chromosome Mapping↗

PubMed1985

Cloning and expression of the pnd gene of R16: determination of transcriptional direction and evolutionary analysis.

The gene promoting nucleic-acid degradation (pnd) of IncB plasmid R16 was cloned into the vector plasmid pACYC177. The pnd gene was found to be located on a 0.55-kilobase (kb) AluI-PstI fragment by constructing subclones carrying various portions of the initially cloned fragment. The direction of transcription of the pnd gene was determined by inserting the gene in both orientations into the lacZ' gene of the plasmid pUR222. In the recombinant plasmid pCM2, transcription of the pnd gene was controlled by the lac promoter region. Addition of cAMP at 42 C resulted in rapid degradation of stable RNA in cells harboring pCM2. In contrast, no RNA degradation was observed in cells harboring pCM14, which has the same insert as pCM2 but in the opposite orientation. The equivalent gene, pnd of IncI alpha plasmid R483, has previously been cloned, and a detailed restriction map of the region has been constructed (Akimoto, S., and Ohnishi, Y. 1982. Microbiol. Immunol. 26: 779-793). We constructed a detailed restriction map of the pnd region of R16 and compared it with that of R483. Restriction analyses revealed a similar structure in these two pnd regions. The results suggest that the pnd genes of R16 and R483 have a common evolutional origin.

Biological Evolution↗

PubMedDate not supplied

[Physical mapping of Streptomyces coelicolar A3(3) actinophages. III. Restriction analysis].

It has been shown that endonucleases HindII, HindIII, SalI and BsuI treatment of phiC62, or phiC43 and phiC31 DNAs forms more than 20 fragments. EcoRI cleaves phiC62, phiC31, phiC31c5 and phiC31c28 into seven fragments, but phiC311yg33 into six fragments. Comparison of molecular weights of DNA restricts obtained after hydrolysis of phage DNAs containing deletions by endonuclease EcoRI made it possible to determine the location of four fragments on restriction map and to orientate this map in relation to the molecule's ends. BamHI cleaves phiC43 DNA into two fragments. By heteroduplexing BamHi site was mapped within the phiC43 insertion sequence.

Bacteriophages↗

PubMed1991

Physical map of the Brucella melitensis 16 M chromosome.

We present the first restriction map of the Brucella melitensis 16 M chromosome obtained by Southern blot hybridization of SpeI, XhoI, and XbaI fragments separated by pulsed-field gel electrophoresis. All restriction fragments (a total of 113) were mapped into an open circle. The main difficulty in mapping involved the exceedingly high number of restriction fragments, as was expected considering the 59% G + C content of the Brucella genome. Several cloned genes were placed on this map, especially rRNA operons which are repeated three times. The size of the B. melitensis chromosome, estimated as 2,600 kb long in a previous study, appeared longer (3,130 kb) by restriction mapping. This restriction map is an initial approach to achieve a genetic map of the Brucella chromosome.

Brucella↗

PubMed1984

A physical and functional analysis of Tn917, a Streptococcus transposon in the Tn3 family that functions in Bacillus.

The erythromycin-resistance (Emr)-conferring transposon Tn917, first isolated in the genus Streptococcus, has in previous work been shown to function efficiently in the spore-forming species Bacillus subtilis, where it has been developed as a tool for identifying and studying sporulation genes. In the present work, a physical analysis of Tn917 was undertaken, including detailed restriction mapping, chemical DNA sequencing, heteroduplex studies, and Southern hybridization analysis, as a first step in understanding the genetic organization of this useful insertion element. The location and transcriptional orientation of the transposon-borne erm gene (the gene responsible for the Emr phenotype) have been determined, and a partial sequence of DNA 5' to the coding sequence of this gene indicates that its inducibility is probably the result of "translational attenuation," a mechanism known to be responsible for the regulation of at least two other gram-positive erm genes. Restriction mapping and heteroduplex analysis have revealed extensive homology between Tn917 and the Staphylococcus transposon Tn551, throughout virtually their entire lengths, and DNA sequencing studies have revealed a remarkably high degree of sequence correspondence within the terminal inverted repeats of Tn917, Tn551 and the gram-negative transposon Tn3. Tn917 was also shown to generate a 5-bp duplication upon insertion, as do Tn3 and Tn551 (and all of the other Tn3-related elements studied thus far), strengthening the conclusion that these three transposons are members of a highly dispersed family of related insertion elements which populate both gram-positive and gram-negative genera.

Bacillus↗

PubMed1982

Ds controlling elements of maize at the shrunken locus are large and dissimilar insertions.

A recombinant clone for the wild-type gene of sucrose synthetase has been selected. Subclones of the genomic clone were made and used as probes to construct a restriction map of the wild-type locus. The transcribed region and the direction of transcription in the map were also determined. Four Ds-induced mutations of the locus that were isolated by B. McClintock were analyzed and partially mapped. Two mutations contain insertions of approximately 20 kb of foreign DNA into the transcribed region; two others are at the 5' end of the gene. There are profound differences in the restriction maps of the Ds elements in the mutants analyzed, even though these elements have a common genetic origin and are separated by only a few generations. Comparison of one pair of closely related Ds elements indicates that these differences may be the result of extensive internal rearrangements. Exchange with other elements in the genome remains an alternative possibility. A revertant of one of the Ds-induced mutations has maintained a 21-22 kb insert at the same location in the transcribed region. This insert differs from its predecessor by extensive rearrangement in two thirds of its length. Part, or all, of this rearrangement presumably permits functional expression of the gene.

Chromosome Mapping↗

PubMed2004

[Analysis of the sequences of the ribosomal DNA ITS2 region for differentiating anopheline mosquitoes within hyrcanus group].

OBJECTIVE: To analyze the genetic characteristics of ribosomal DNA ITS2 region for differentiating anopheline mosquitoes within Hyrcanus group. METHODS: The ribosomal DNA ITS2 region of both laboratory line and filed collected An. anthropophagus and An. sinensis as well as the field collected An. yatsushiroensis and An. lesteri were amplified and sequenced. The sequencing data were then analyzed for the restriction mapping using Omega Sequencing analysis program. RESULTS AND CONCLUSION: The length of the sequences of An. sinensis, An. anthropophagus, An. yatsushiroensis and An. lesteri are 472, 452, 456 and 456 bp respectively. The restriction mapping showed that there were different restriction digesting sites among the ribosomal DNA ITS2 region sequences from An. sinensis, An. anthropophagus, An. yatsushiroensis and An. lesteri. On the basis of the sequence differences among the anopheline species within Hyrcanus group, it is possible to develop new technique for genetic identification of anopheline mosquitoes.

Animals↗

PubMed1993

The human complement component C8B gene: structure and phylogenetic relationship.

The eighth component of human complement (C8) is a serum protein that consists of three chains (alpha, beta and gamma), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the beta-subunit is non-covalently bound to the disulfide-linked alpha-gamma subunit. Using a full-length C8 beta cDNA probe, we isolated several clones from human genomic lambda DNA libraries. Four lambda clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were "shotgun" subcloned into M13. C8 beta-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. By using polymerase chain reaction (PCR) primers located in the adjacent intron sequences, all 12 exons of the C8B gene could be amplified from genomic DNA. All fragments showed the expected sizes. The sizes of eight introns could be determined by using primer pairs that amplified two exons and the enclosed intron, and by restriction mapping. These analyses and the insert sizes of the genomic lambda clones indicate that the C8B gene has a total size of approximately 40 kb. The polymorphic TaqI site of the C8B gene localized in intron 11 could be demonstrated by direct restriction fragment analysis of a PCR fragment containing exons 11 and 12, and the enclosed intron 11. Homology comparison of the C8B gene with C8A and C9 on the basis of the exon structure confirmed the ancestral relationship known from the protein level.

Base Sequence↗

PubMed1992

Similarity between the group B and A streptococcal C5a peptidase genes.

Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.

Adhesins, Bacterial↗

PubMed1994

[Relationship between gut origin bacteria and wound infection after thermal injury].

The pUC19 plasmid vector trace with restriction map analysis and fluorescence labelling bacteria method were applied to study the relationship between the gut origin bacteria and wound infection. According to the characteristic of pUC19 plasmid, a special animal model was designed. 110 Wistar rats received 30% TBSA full thickness burns. On hours 6, 12, 24, 48 and day 12 postburn, injured animal were killed. Subeschar tissue homogenates were examined under fluorescence microscope, and bacterial culture, isolation of plasmids and restriction map analysis were also carried out. The results show that during early stage of burns, 32.5% of fluorescence labelling bacteria and 10.81% of pUC19 plasmid vectors could be detected from the subeschar specimens. 12 day postburn, the detectable rage of pUC19 plasmid vector increased to 62.5%. Beside the factor of early colonization, the contaminative route form gut perineum and then wounds should be considered.

Animals↗

PubMed1993

Fine mapping of the FSHD gene region orientates the rearranged fragment detected by the probe p13E-11.

We have produced a fine restriction map around the locus D4F104S1 (previously designated D4S810); a probe to this locus, p13E-11, identifies a polymorphic EcoRI fragment containing 3.2kb tandem repeats and detects DNA rearrangements associated with facioscapulohumeral muscular dystrophy (FSHD). We developed an STS (D4F106S1) which maps 2kb proximal to D4F104S1, and used this to isolate a 470kb YAC (y25C2E) from the ICI YAC library and a 930kb YAC (y956A11) from the CEPH megabase library. Both YACs contain the loci D4S139, D4F35S1 and D4F104S1. A cosmid library was produced from YAC y25C2E and two cosmid contigs constructed; a 115kb contig encompassing D4S139, and one of 135kb linking D4F35S1 and D4F104S1 and extending distal to the EcoRI fragment detected by p13E-11. A fine restriction map of both these contigs has been generated, allowing the orientation of the EcoRI fragment rearranged in FSHD to be determined. YAC y956A11 was used to confirm the integrity of y25C2E and the map of this region. 9B6A, a probe to the homeobox region of the tandem repeat D4Z4, identified a cross-hybridising sequence proximal to D4F104S1, however, p13E-11 does not detect this additional locus. CpG islands were identified between D4S139 and D4F35S1 and within each copy of the tandem repeat. The probe 9B6A detected each copy of the repeat motif, suggesting there is homeobox present in every copy of the 3.2kb repeat.

Base Sequence↗

PubMed1989

Cloning of the crystalline cell wall protein gene of Bacillus licheniformis NM 105.

A protein with a tetragonal pattern, defined as RS protein, was found on the wall surface of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C. The protein was present on the wall surface of the exponential-growth-phase cells, but at the stationary growth phase it was overproduced and hypersecreted. This protein was precipitated to homogeneity from the culture fluid by 80% ammonium sulfate saturation and chilled acetone. The molecular mass of the protein was 98 kilodaltons, and it had a single subunit in a sodium dodecyl sulfate gel. Specific anti-RS antibody was generated in rabbits and used to immunolabel the RS protein on the cells at different growth phases. In early-exponential-growth-phase cells, the outside surface of the wall, the cytoplasm, and the inside surface of the cytoplasmic membrane were labeled. In stationary-growth-phase cells, the cytoplasm was poorly labeled, but the labeling on the outside surface of the wall was high. AB. licheniformis NM 105 gene library was made by using the lambda phage EMBL3. The RS protein expression from this gene library was detected by a modified autoradiographic procedure. One of the amplified RS protein-positive plaques (4213-1) containing recombinant DNA was chosen, and the restriction map of this DNA was prepared. The RS protein expressed in Escherichia coli NM 539 infected with 4213-1 recombinant phage had a lower molecular mass than the purified authentic RS protein. The 4.5-kilobase-pair (kbp) SalI-EcoRI fragment of the recombinant DNA was cloned in the shuttle plasmid pMK4 to construct pMK462, which was expressed in B. subtilis MI112 and produced the RS protein identical in molecular mass to the purified authentic RS protein. The RS protein expression was also demonstrated in cryosections of transformed E. coli and B. subtilis cells by immunoelectron microscopy. The 1.2-kbp SalI-HindIII and 1.8-kbp HindIII-HindIII recombinant DNA restriction enzyme fragments, respectively, from the right of the restriction map produced anti-RS antibody cross-reacting proteins. The expression of the 1.2-kbp SalI-HindIII DNA fragment cloned in pUC8 could be induced with isopropyl-beta-D-thiogalactopyranoside. The 1.8-kbp DNA restriction fragment hybridized with both the chromosomal DNA of strain NM 105 and the recombinant phage 4213-1 DNA. The RS gene expression was finally demonstrated in transformed E. coli 539 cells by in situ hybridization of frozen thin sections with the 1.8-kbp HindIII biotin-dATP probe and immunolabeling these with anti-biotin immunoglobulin G and protein A-gold.

Bacillus↗

PubMed1995

Genetic mapping of restriction landmark genomic scanning loci in the mouse.

Restriction landmark genomic scanning (RLGS) was originally proposed as a high-speed method for surveying a large number of restriction landmarks in genomic DNA. The effort to apply this method to genetic analysis has been made, resulting in developing the new approach for the rapid construction of the genetic map of complex mammalian genomes (RLGS spot mapping). Especially, the use of NotI as the restriction landmark for genetic studies suggests that there is a high probability that a significant number of these RLGS loci will be associated with CpG islands of functional genes. Moreover, it is possible to use the RLGS spot mapping to analyze genetic map-poor species very rapidly for linkage of recessive mutations or segregating traits, because it does not rely upon cloned probes or sequences. In this paper, we summarize the progress that has been made in the practical application of the RLGS method to genetic analysis using congenic strains, recombinant inbred (RI) strains, and in interspecific backcrosses of mice.

Animals↗