Generation of 28 sequence-tagged sites (STSs) from yeast artificial chromosome (YAC) clones anchored at human chromosome 21q22.1 region.

Twenty-eight sequence-tagged sites (STSs) were newly generated from the DNA sequences of vector-insert junctions from yeast artificial chromosomes (YACs) anchored at chromosome 21q22.1 region. The insert DNAs adjacent to vector arms were specifically amplified through inverse PCR method to clone into pUC19 vector for sequencing. Sixty DNA junctions from 44 CEPH YAC clones were cloned and sequenced. Of these DNA sequences of junctions between vector-arms and DNA inserts, twenty-eight STSs were finally obtained to show the accurate amplification, which is specific for human chromosome 21. The sets of 28 STSs were useful to build fine YAC contigs by STS-mediated YAC walking at the 21q22.1 region.

1.8-megabases fine physical map encompassing IFNAR and AML1 loci on human chromosome 21q22.1.

A long-range restriction map of the 1.8-megabases (mb) region encompassing the area between the interferon-alpha receptor and the acute myelogenous leukemia loci on human chromosome 21q22.1 was constructed after analysis of both the contiguous yeast artificial chromosome (YAC) clones and genomic DNA. Analysis of pulsed-field gel electrophoresis of lymphoblastoid DNA digested with three rare-cutting enzymes, Not I, Mlu I, and Nru I, revealed the positions of 17 markers on each restriction map. The 1.8-mb YAC contig that covers this region was obtained through YAC walking mediated by sequence-tagged sites (STSs), with 29 STSs including 12 newly generated YAC end-specific STSs. The consensus restriction map from 15 overlapping YACs and the positioning of the STS markers on each clone allowed 24 markers including 4 Not I-linking STSs to be ordered and mapped physically. Comparison of the maps revealed that the proximal region contains more unmethylated CpG islands than the distal region, which suggests that many expressed genes are in the proximal region. This fine consensus physical map will be informative and useful for construction of contigs of cosmid, P1, or BAC clones for further large-scale sequencing in this gene-rich region.

[Quantitative evaluation of nystagmus by an image-analysis system].

PURPOSE: We attempted to apply a newly developed image-analysis system for measurement and analysis of nystagmus. METHOD: Eye movements were recorded by digital video through a head-mounted charge coupled device (CCD) camera. The recorded movie was converted into black and white in order to detect the area of the pupil. Horizontal and vertical eye positions were determined by calculating the centroid of the pupil. Torsional angle was calculated using the iris striate pattern around the pupillary margin. RESULTS: The parameters (amplitude, cycle, etc.) of nystagmus were calculated easily by the new image-analysis system from the recorded images. As examples, the foveation period was measured accurately in a case of jerky-type congenital nystagmus. Very regular cycles of intorsional attack period were revealed in a case of superior oblique myokymia. A case of cork-screw-like nystagmus showed a characteristic combination of large and small cycles unassociated with torsion. CONCLUSION: This image-analysis system was useful for quantitative analysis of nystagmus, and especially for measurement of torsion. Detailed waveforms and specific rhythms of nystagmus, which could not be recognized by observation, were demonstrated by this system.

[Primula dermatitis].

Ten patients with primula dermatitis observed in Hokkaido were reported. All patients presented positive patch test reactions to a part of an extract of primula obconica. Postlesional pigmentation is prominent in patients with recurrent dermatitis. Histologic features were liquefaction degeneration of a basal layer of the epidermis and incontinentia pigmenti histological, i.e., lichenoid tissue reaction.

Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis.

Helicase-related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase-related proteins make up several gene families. In this study, we have identified 134 ORFs that encode helicase-like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase-related proteins. Multiple alignment of the ORF sequences resulted in the 134 ORFs being classified to 11 clusters. Seven out of 21 previously uncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) were identified by systematic gene disruption, to be essential for vegetative growth. Three (YDR332w, YGL064c, and YOL095c) out of the remaining 14 dispensable ORFs exhibited the slow-growth phenotype at 30 degrees C and 37 degrees C. Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growth conditions by Northern analysis and reverse transcription-polymerase chain reaction, indicating that all of the 43 tested ORFs were transcribed. Interestingly, we found that the level of transcript from 34 helicase-like genes was markedly increased by heat shock. This suggests that helicase-like genes may be involved in the biosynthesis of nucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for the repressed synthesis of mRNA and protein.

Sarcoidosis presenting as a scarring alopecia: report of a rare cutaneous manifestation of systemic sarcoidosis.

We report a 51-year-old woman with alopecia caused by sarcoidosis. The lesion enlarged within 4 years and only repeated biopsies enabled the diagnosis. The medical work-up revealed that the patient had asymptomatic pulmonary involvement. Scarring alopecia is a rare complication of sarcoidosis and biopsy from the active margin may lead to the diagnosis.

Mapping of the 70 kDa, 34 kDa, and 11 kDa subunit genes of the human multimeric single-stranded DNA binding protein (hSSB/RPA) to chromosome bands 17p13, 1p35-p36.1, and 7p21-p22.

Human single-stranded DNA binding protein (hSSB/RPA) is a multimeric single-stranded DNA binding protein consisting of three subunits of 70 kDa, 34 kDa, and 11 kDa. Human SSB was isolated from HeLa cells as an essential factor for the in vitro replication of simian virus 40 DNA. We and others have isolated and sequenced cDNAs for each subunit of the SSB. The chromosome on which each gene is located was determined through the analysis of a panel of human/hamster somatic cell hybrids using the polymerase chain reaction with pairs of synthetic oligonucleotide primers from the 3'-untranslated sequences of the genes. Genomic clones for each gene were isolated from a genomic cosmid library prepared from human lymphoblastoid cells. Using those clones as probes, we have carried out fluorescence in situ hybridization to human metaphase chromosomes and have mapped the 70 kDa subunit gene to 17p13, the 34 kDa subunit gene to 1p35-p36.1, and the 11 kDa subunit gene to 7p21-p22. Since hSSB participates in replication, recombination and repair of DNA, the physical mapping of hSSB genes may aid in the identification of human hereditary diseases associated with aberrant DNA reactions caused by genetic alterations of the hSSB.

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23.

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Assignment of the 49-kDa (PRIM1) and 58-kDa (PRIM2A and PRIM2B) subunit genes of the human DNA primase to chromosome bands 1q44 and 6p11.1-p12.

DNA primase is an essential replication protein that catalyzes the synthesis of oligoribonucleotide primers. DNA primase, consisting of two subunits (p49 and p58), plays a key role in both the initiation of DNA replication and the synthesis of Okazaki fragments for lagging strand synthesis. We mapped the locations of human chromosomes of the genes coding for both subunits [p49 (PRIM1) and p58 (PRIM2)] by PCR amplification using DNAs of a panel of somatic hybrids, to chromosomes 1 and 6, respectively. The PRIM1 gene was mapped to 1q44, and two PRIM2 loci (PRIM2A and PRIM2B) were detected at 6p11.1-p12 by fluorescence in situ hybridization using several genomic DNA probes.

A long-range physical map of human chromosome 21q22.1 band from the YAC continuum.

The human Chromosome (Chr) 21q22.1 region contains several genes for cytokines and neurotransmitters and the gene for superoxide dismutase (mutant forms of which can cause familial amyotrophic lateral sclerosis). A region of approximately 5.8 Mb encompassing D21S82 and the glycinamide ribonucleotide transformylase (GART) loci was covered by overlapping YAC clones, which were contiguously ordered by clone walking with sequence-tagged site (STSs). A total of 76 markers, including 29 YAC end-specific STSs, were unambiguously ordered in this 5.8-Mb region, and the average interval between markers was 76 kb. Restriction maps of the YAC clones with rare-cutting enzymes were simultaneously prepared, and the restriction sites were aligned to obtain a consensus restriction map of the proximal region of the 21q22.1 band. The restriction map made from 44 overlapping YACs contains 54 physically assigned STSs. By integrating the consensus map of the adjacent 1.8-Mb region, we obtained a fine physical map spanning 6.5 Mb of human Chr 21q22.1. This map contains 24 precisely positioned end-specific STSs and 12 NotI-linking markers. More than 39 potential CpG islands were identified in this region and were found to be unevenly distributed. This physical map and the YACs should be useful as a reference map and as a resource for further structural analysis of the Giemsa-negative band (R-band) of Chr 21q22.1.

Quantitative Evaluation of Nystagmus by an Image-analysis System.

Purpose: We attempted to apply a newly developed image-analysis system for measurement and analysis of nystagmus.Method: Eye movements were recorded by digital video through a head-mounted charge coupled device (CCD) camera. The recorded movie was converted into black and white in order to detect the area of the pupil. Horizontal and vertical eye positions were determined by calculating the centroid of the pupil. Torsional angle was calculated using the iris striate pattern around the pupillary margin.Results: The parameters (amplitude, cycle, etc.) of nystagmus were calculated easily by the new image-analysis system from the recorded images. As examples, the foveation period was measured accurately in a case of jerky-type congenital nystagmus. Very regular cycles of intorsional attack period were revealed in a case of superior oblique myokymia. A case of cork-screw-like nystagmus showed a characteristic combination of large and small cycles unassociated with torsion.Conclusion: This image-analysis system was useful for quantitative analysis of nystagmus, and especially for measurement of torsion. Detailed waveforms and specific rhythms of nystagmus, which could not be recognized by observation, were demonstrated by this system.