Yoshiyuki Sakaki

Dideoxy sequencing method using denatured plasmid templates.

The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved. The method is simple and rapid: the recombinant plasmid DNA is extracted and purified by rapid alkaline lysis followed by ribonuclease treatment. The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers. It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction. We examined each step of the procedure, and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA; (ii) a heptadecamer rather than a pentadecamer is recommended as a primer; and (iii) the sequencing reaction should be done at 37 degrees C or higher rather than at room temperature. The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA.

Genomic organization of the human amyloid beta-protein precursor gene

Abstract Amyloid β-protein (BP) deposited in Alzheimer brains is a cleavage product of a larger precursor (BPP). The BPP gene encodes three types of mRNA generated by alternative splicing, two of which contain the sequence encoding Kunitz-type serine-protease inhibitor (serpin). To investigate the regulatory mechanisms of BPP synthesis at the gene level, we isolated 36 genomic DNA clones covering all the exons of the human BPP gene. This gene consists of 18 exons and spans more than 170 kb. BP is encoded by the 16th and the 17th exons and the serpin domain by the 7th exon. Sequence analysis showed that the 7th and 8th introns lack a typical branchpoint for splicing. This might relate to the alternative splicing. The promoter of the BPP gene has some characteristics of those of housekeeping genes and contains a number of possible methylation sites. The methylation status of the promoter was analyzed by Southern blotting but no alteration was observed among tissues and between control and Alzheimer brains. We also tested the roles of two possible activator protein-1-binding sites and a possible heat-shock element found within the promoter. Northern blotting showed that the transcription of the BPP gene was apparently induced by 12-O- tetradecanoylphorbol -13- acetate (phorbol derivative) in HeLa cells.

Pro→Leu change at position 102 of prinon protein is the most common but not the sole mutation related to Gerstmann-Sträussler syndrome

The host-encoded prion protein (PrP) is a component of transmissible amyloid deposited in the brains affected by Gerstmann-Sträussler syndrome (GSS). Recently GSS in two unrelated Caucasian families has been reported to be linked to an amino acid change in PrP codon 102, proline to leucine (Leu102). However, it has not been clear whether the change is commonly found to GSS regardless of ethnic origin. We report here that Leu102 is also found in all the Japanese GSS patients tested. Interestingly, one French GSS patient was found to have another change, alanine to valine in codon 117 (Val117), instead of Leu102. Our results indicate that Leu102 is closely related to GSS irrespective of ethnic origin, but not the sole mutation related to GSS. Val117 may also be related to GSS.

SNP and haplotype mapping for genetic analysis in the rat.

The laboratory rat is one of the most extensively studied model organisms. Inbred laboratory rat strains originated from limited Rattus norvegicus founder populations, and the inherited genetic variation provides an excellent resource for the correlation of genotype to phenotype. Here, we report a survey of genetic variation based on almost 3 million newly identified SNPs. We obtained accurate and complete genotypes for a subset of 20,238 SNPs across 167 distinct inbred rat strains, two rat recombinant inbred panels and an F2 intercross. Using 81% of these SNPs, we constructed high-density genetic maps, creating a large dataset of fully characterized SNPs for disease gene mapping. Our data characterize the population structure and illustrate the degree of linkage disequilibrium. We provide a detailed SNP map and demonstrate its utility for mapping of quantitative trait loci. This community resource is openly available and augments the genetic tools for this workhorse of physiological studies.

Complementary DNA for the mouse homolog of the human amyloid beta protein precursor

The human amyloid beta protein is a major component of brain amyloid found in patients with Alzheimers disease. As an initial step to understand the biological function of its precursor protein, we have isolated cDNA for the mouse homolog of the human beta protein precursor. Comparison of the predicted amino acid sequence with that of human revealed a quite high degree of homology (96.8%), and the calculated evolutionary rate of the mRNA at amino acid substitution site was relatively low (0.1 x 10(-9)/site/year). The mRNA was abundant in brain and kidney, and also detected in other tissues at low level. These results indicated that this protein is highly conserved through mammalian evolution and may be involved in a basic biological process(es).

The 717Val→Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups

Alzheimers disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.

Gly145 to Arg substitution in HBs antigen of immune escape mutant of hepatitis B virus.

A Japanese child born to an HBeAg-positive carrier mother received anti-HBs immunoglobulins and a plasma-derived HBs vaccine with a poor anti-HBs-antibody response. The child, who is now 3 years old, is presently suffering from chronic hepatitis with unusual serological findings that are positive for HBsAg, anti-HBs and HBeAg, since being infected with a measles virus at 12 months of age. The nucleotide sequences of the S region of HBV DNA obtained from the patient, the mother and an HBeAg-positive brother were completely identical except for one nucleotide at position 587 (mother and brother: guanosine, patient: adenosine), giving an amino acid change: Gly - greater than Arg at position 145 of the major HBs protein.

Nucleotide sequences and unusual electrophoretic behavior of the W chromosome specific repeating DNA units of the domestic fowl, Gallus gallus domesticus

Nucleotide sequences of three independently cloned repeating units of the W chromosome-specific repettive DNA sequences (“XhoI family”) of the chicken were determined. All three units are 717 bp long with XhoI sites at both ends. There are only 21 sites out of 717 bases where a single base change occurs in one of the three clones. Each of these repeating units consists of 34 tandem repeats of about 21 bp. Sequences of some members of these internal repeats are not well conserved, but the majority of the repeats are characterized by the presence of (A)3–5 and (T)3–5 clusters separated by 6–7 relatively G+C-rich base pairs. One striking feature of the cloned 717 bp repeating units is that they migrate unusually slowly on electrophoresis in polyacrylamide gels. The same feature is also shown by a genomic population of the 0.7 kb repeating units recovered from XhoI digests of the genomic DNA of the female chicken. This anomalous behavior is attributed to the occurrence of DNA curvatures because of the above sequence characteristics and partial recovery of the electrophoretic mobility in the presence of distamycin A. Another feature of the 717 bp repeating unit is the presence of 438 and 159 nucleotide-long open reading frames (ORFs) at each end of the unit. A possible function of the XhoI family sequences in the heterochromatization of the W chromosome and the significance of the ORFs are discussed.

Diagnosis of Familial Amyloidotic Polyneuropathy by Recombinant DNA Techniques

An amino acid substitution of Met for Val at position 30 of plasma prealbumin is known to be closely related to heredo-familial amyloidotic polyneuropathy(FAP). As a first step in development of a direct method for diagnosis of the disease, cDNA for normal human prealbumin was cloned and its nucleotide sequence was determined. Our results showed that the nucleotide substitution responsible for the Val----Met change results in formation of new restriction sites for BalI and NsiI. By Southern blot hybridization analysis, the expected restriction sites were actually detected in the prealbumin locus of patients. Thus, a method was developed for diagnosis of the disease presymptomatically and prenatally.

Immunochemical, molecular genetic, and transmission studies on a case of Gerstmann‐Straussler‐Scheinker syndrome

Using immunostaining with anti-prion protein (PrP) antiserum, we detected numerous kuru plaques in the brain of a 24-year-old man with Gerstmann-Straussler-Scheinker syndrome. Immunoreactivity on Western blotting of the protease-resistant PrP fraction from the frozen brain was weak. PrP gene analysis showed substitution of alanine to valine in codon 117 but no substitution in codon 102. As the experimental transmission of the disease to mice was negative, a pathogen of a relatively low infectivity may cause the disease in predisposed family members.