Five age-dependently expressed genes in mouse brain revealed by the fluorescence differential display-PCR technique.

We used a fluorescence differential display-PCR (FDD-PCR) technique to analyze the genes expressed in mouse brains collected at nine different developmental stages ranging from 3 days to 15 months after birth, and 5 age-dependently expressed genes were found. Age-dependent expression of each of these 5 genes was confirmed by quantitative real-time PCR analysis. Of the 5 genes, 4 (B1-B4) had high homology with the nucleotide sequences of cDNA clones of known mouse genes (myelin proteolipid protein, transferrin, embryo cDNA from the RIKEN full-length enriched library, and protein tyrosine phosphatase), and the rest (B5) with expressed sequence tags of an unknown gene. Sequencing analysis of the full-length cDNA constructed based on the B5 sequence demonstrated that the gene product of B5 was identical to G-substrate, a specific substrate for cGMP-dependent protein kinase. The expression patterns of known genes obtained in our study may provide a further opportunity to investigate the biological and physiological roles of the proteins they encode.

Characterization and haplotype analysis of the polymorphic Y-STRs DYS443, DYS444 and DYS445 in a Japanese population.

From sequence database information we have newly identified three male-specific and polymorphic tetranucleotide STRs, DYS443 (GDB: 10807127), DYS444 (GDB: 10807128) and DYS445 (GDB: 10807129) on the Y chromosome. Analysis of 190 Japanese males revealed 6, 5 and 4 alleles in the DYS443, DYS444 and DYS445 systems, with calculated STR diversities of 0.68, 0.57 and 0.53, respectively. The cumulative haplotype diversity of the five Y-STRs DYS441, DYS442, DYS443, DYS444 and DYS445 was calculated to be 0.95 and therefore application of these STRs may yield very useful information for forensic individualization.

Identification and characterization of two novel human polymorphic STRs on the Y chromosome.

From sequence database information, we have identified two male-specific and polymorphic tetranucleotide STRs, DYS 441 (GDB:10013873) and DYS 442 (GDB: 10030304), on the Y chromosome. Analysis of 184 males allowed 7 and 5 alleles to be distinguished in the DYS 441 and DYS 442 systems, respectively, yielding 21 haplotypes. The gene diversities were 0.72 and 0.51, respectively and the haplotype diversity was 0.85.

Cloning, mapping, genomic organization, and expression of mouse M-LP, a new member of the peroxisomal membrane protein Mpv17 domain family.

We have identified a mouse full-length cDNA and gene encoding a novel protein (M-LP), based on an expressed sequence tag (EST) sequence (GenBank Accession No. AI482564) obtained by differential display screening of age-dependently expressed genes in mouse kidney. The ML-P gene is composed of three exons, ranges over 5 kb on mouse chromosome 16B1-B2 and is expressed as two transcripts (1455 and 3058 bp), both of which include the same open-reading frame encoding 194 amino acids. M-LP is expressed mainly in kidney and spleen and shows age-dependent expression. M-LP has sequence homologies and membrane topologies very similar to the Mpv17 protein, a peroxisomal protein involved in the development of early-onset glomerulosclerosis. Search of the protein domain family database (ProDom) revealed that M-LP is a new member of the Mpv17 domain family (PD008400).

Age-related changes of gene expression in mouse kidney: fluorescence differential display--PCR analyses.

We used a fluorescence differential display--PCR (FDD-PCR) technique to analyze the genes expressed in mouse kidneys collected at nine different developmental stages ranging from 3 days to 15 months after birth. We found ten genes that were age-dependent and differentially-expressed in the kidneys during our experimental period. We confirmed by comparative RT-PCR that of the ten cDNAs, seven showed reproducible age-dependent expression. Four of the nucleotide sequences of these cDNA clones, had high homology with known genes (fibronectin, soluble guanylyl cyclase alpha-1 subunit, cytosolic aldehyde dehydrogenase and mitochondrial DNA), and three with expressed sequence tags of unknown genes. The FDD-PCR method was very useful for detecting new age-related genes expressed differentially in the mouse kidney.

Deoxyribonuclease I phenotyping from saliva stains.

Good typing results were obtained using a newly developed method for extraction and purification of deoxyribonuclease I (DNase I) from saliva stains. Previously, DNase I phenotyping from saliva stains has been unsuccessful because of low enzyme activity and heavy contamination. Salivary DNase I was extracted from stains using phosphate buffer containing Nonidet P-40. Extracts were purified using Phenyl Sepharose CL-4B gel. Electrophoresis was performed, and DNase I was successfully phenotyped. All of the DNase I phenotypes, which were obtained from saliva stains using this new method, were identical to the phenotypes determined from urine samples. Moreover, DNase I was correctly phenotyped from saliva stains that had been stored for over three months at room temperature or at 37 degrees C. These results suggest that DNase I polymorphisms provide valuable information for forensic characterization of saliva stains.

Detection of isozymes of deoxyribonucleases I and II on electrophoresed gels with picogram sensitivity using SYBR Green I.

A highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32- to 128-fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 microL human serum. This new technique has been named SG-DAFO, after its original dried agarose film overlay method using EB (EB-DAFO).

DYS19 marker in Japanese populations.

Allele frequencies of the Y chromosome-specific short tandem repeat system DYS19 (Y-27H39) were determined from blood samples obtained from 251 unrelated Japanese in Fukui and Gunma Prefectures. Five alleles that contained 10, 11, 12, 13 and 14 repeats of GATA were detected. Allele frequency distributions in Japanese populations were different from those in other Asian and Caucasian populations.

The fifth allele of the human deoxyribonuclease I (DNase I) polymorphism.

The fifth allele, DNASE1*5, of human deoxyribonuclease I (DNase I) has been discovered. Polymerase chain reaction fragments containing exon 5 of the DNase I gene were screened for DNA polymorphism using single-strand conformation polymorphism (SSCP) analysis. DNAs from 114 unrelated Japanese and 81 German individuals were tested and a new variant was detected. By DNA sequencing analysis, this variant was found to be caused by a heterozygous G-A transition at nucleotide position 1227 that results in a Val to Met substitution at amino acid position 92 of the mature enzyme. The nucleotide substitution was also confirmed by mismatched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. Genotyping of the variant could be carried out by three independent reactions based on PCR amplification, and phenotyping by isoelectric focusing followed by immunostaining. The results supported the presence of the fifth codominant allele, DNASE1*5, which generates a new isozyme.

Identification of the nucleotide substitution that generates the fourth polymorphic site in human deoxyribonuclease I (DNase I).

In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1-2, and 2.

Alpha-2-HS-glycoprotein (AHSG) polymorphism in semen and saliva.

Polymorphism of alpha-2-HS-glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin-layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P-40 into the IEF gel (pH 4.2-4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2-5 microL semen and 50-100 microL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.

Effect of iodopyracet on renal excretion of sulfamethizole in rabbits.

To predict quantitatively drug interaction kinetics from the single-drug clearance studies, we examined the effect of iodopyracet (IOD) on sulfamethizole (SMZ) excretion in rabbits. Even though the decline of systemic IOD plasma concentration was linear, the renal clearance of SMZ decreased significantly in the presence of IOD. The results could be described by a perfusion model incorporated with the competitive inhibition for tubular secretion. For IOD with a high extraction ratio, it was suggested that a heavy load of the drug was supplied to the sites of secretion and caused the saturation of transport systems, even though the renal excretion kinetics were apparently linear in respect to the systemic circulation. These facts indicated that a linear relationship between the concentrations in the systemic circulation and at the sites of tubular secretion can not always be presumed. Consequently, SMZ-IOD interaction study stressed the importance of the drug concentrations at the sites of interaction for quantitative elucidation of drug-drug interactions.