Molecular analysis of human Chromosome 16 cosmid clones containing NotI sites

To test the feasibility of using cloned NotI sites as markers for physical mapping, we have screened for cosmid clones spanning the NotI sites on human Chromosome (Chr) 16. Fluorescence in situ hybridization analysis of these clones confirms the previously reported cluster of NotI sites on 16p13.3. Methylation status of the cloned NotI sites on genomic DNA was established by hybridization of the cosmids to Southern blots containing EcoRI and EcoRI/NotI digest of genomic DNA. These results indicated that four of six clones included in our study can be used as linking clones for physical mapping. Two clones have NotI sites which are not cleavable in the cell lines tested. In one clone, the NotI site exists as an isolated rare-cutting restriction enzyme site, whereas in the other clone the NotI site appears to be island-related.     

Localization of the NotI clones from human chromosome 3 on quail microchromosomes

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.     

Isolation and regional mapping of NotI and EagI clones from human chromosome 21

NotI and EagI boundary libraries were constructed for human chromosome 21. One hundred forty-seven clones were isolated from the somatic cell hybrid 72532X-6 and localized using a hybrid mapping panel. After identification of those clones, which were isolated more than once, as well as those probes derived from a previously unrecognized integrated non-chromosome-21 fragment, 58 individual boundary clones (plus 2 additional NotI-EcoRI clones isolated from a flow-sorted library) were localized to 11 separate regions. The distribution of these probes is highly nonrandom, with 50% of the clones located in the distal band 21q22.3. Two probes, Not50 and Eag101, map to regions in the very proximal long arm which may contain the gene responsible for familial Alzheimer's disease (AD1), and Not50 would appear to be more proximal than D21S16 (E9). Twenty-eight probes map to the region between superoxide dismutase (SOD1) and the ETS2 oncogene, which appears to contain genes responsible for many of the phenotypic features of Down syndrome. Twenty clones contain (GT)n repeats, as determined by hybridization to a CA polymer, and should provide additional highly polymorphic probes. Closure of gaps in the physical linkage map of chromosome 21 should be facilitated by the isolation of these probes, as they identify many of the unmethylated CpG-rich islands that have hindered pulsed-field gel analysis. They will also be useful in identifying a set of genes in proximity to NotI and EagI restriction sites, as well as conserved DNA sequences for comparative mapping studies.     

NotI flanking sequences: a tool for gene discovery and verification of the human genome

A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera’s database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity ≥90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000–20 000 NotI sites, of which 6000–9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.     

A set of BAC clones spanning the human genome

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.     

Construction of NotI restriction map of the Streptococcus mutans genome

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.     

Images of Alu-sequence in 7 DNA clones from the human genome

Information theory methods were used for computer search of Alu-like sequences in human DNA and RNA. Eight new regions related to the Alu repeat sequence was revealed in 85 clones from the EMBL-5 data bank. Some of these regions are purine-pyrimidine images of Alu repeats sequence, the rest are more complex images of Alu repeat sequence. A new definition for the likeness of different sequences--information image of sequence--was introduced. This information theory application greatly increases the power of DNA sequences computer analysis.     

Construction and regional localization of clones from a NotI linking library from human chromosome 17q

A NotI linking library was constructed from a somatic cell hybrid containing chromosome 17q as its only human material. A total of 112 human clones were assigned to nine regions of 17q using a somatic cell hybrid mapping panel. The library includes clones that detect the acute promyelocytic leukemia and von Recklinghausen neurofibromatosis translocation breakpoints at 17q11.2-12 and 17q11.2, respectively, on pulsed-field gel electrophoresis. The mapped clones represent over 50% of the estimated number of NotI sites on 17q, and therefore constitute an important resource for long-distance mapping.     

Saturating the region of the polycystic kidney disease gene with NotI linking clones.

A NotI-linking library was constructed from a radiation hybrid containing fragments of human chromosome 16. The clones were mapped on a panel of somatic cell hybrids, and 10 different NotI site-containing clones were localized close to and between genetic markers flanking the PKD1 locus. With pulsed-field gel analysis the clones were shown to be distributed over four adjacent ClaI fragments covering 1,200 kb.     

The genome of Haemophilus influenzae Rd has a unique NotI site

Previous analysis of physical maps of Haemophilus influenzae, which is circular and 1.9 Mb in length [Lee and Smith, J. Bacteriol. 170 (1988) 4402-4405; Kauc et al., J. Bacteriol. 171 (1989) 2474-2479], did not detect any NotI (GCGGCCGC) restriction sites. A transposon, Tn916, was constructed to contain a NotI linker cloned into its NciI site and introduced into the H. influenzae chromosome. NotI digestion of chromosomes containing a Tn916-associated NotI site followed by separation of fragments by field-inversion gel electrophoresis revealed the presence of two fragments obtained by two NotI cuts, one in Tn916 and the other, a unique, 'natural' NotI site in the original chromosomal DNA. The examination of other Haemophilus strains demonstrated the presence of one or more NotI sites in all of those tested.     

Large-scale analysis of pseudogenes in the human genome

Pseudogenes are considered as genomic fossils: disabled copies of functional genes that were once active in the ancient genome. Recently, whole-genome computational approaches have revealed thousands of pseudogenes in the genomes of the human and other eukaryotes. Identification of these pseudogenes can improve the accuracy of gene annotation. It also offers new insight on the evolutionary history and the stability of the genome as a whole.     

A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC

We demonstrate that micro-dissection can be used for isolating NotI linking clones from the human 3p21-pter region. This approach is an improvement to positional cloning techniques, since NotI linking clones are directly linked with genes.     

Human chromosome 3: integration of 60 NotI clones into a physical and gene map

Sequence tagged sites generated for 60 NotI clones (NotI-STSs) from human chromosome 3-specific NotI-jumping and NotI-linking libraries were physically located using PCR screening of a radiation hybrid (RH) GeneBridge4 panel. The NotI map of chromosome 3 was generated using these RH-mapping data and those obtained earlier by FISH and sequencing of the corresponding NotI clones. The sequences of the NotI clones showed significant homologies with known genes and/or ESTs for 58 NotI-STSs (97%). These 58 NotI clones displayed 91-100% identity to 54 genes and 23 cDNA/EST clones. One known and two hypothetical protein-coding genes were localized for the first time and nine cDNA clones (unknown genes) were also carefully mapped only in this work. Three newly mapped genes are histone gene H1X (NR1-BK20C) and genes for hypothetical proteins THC1032178 and THC1024604 (NL1-243).     

Computational analysis of human genome polymorphism

While genome-era technologies focused on complete genome sequencing in various organisms, post-genome technologies aim at the understanding of the mechanisms of genetic information processing and elucidation of within-species variation. Single nucleotide polymorphisms (SNPs) are the most common source of genome variation in the human population. Nonsynonymous SNPs that occur in coding gene regions and result in amino acid substitutions are of particular interest. It is thought that such SNPs are responsible for phenotypic variation, quantitative traits, and the etiology of common diseases. PolyPhen is a computational tool for the prediction of putatively functional nonsynonymous SNPs by combining information of various types. The application areas of PolyPhen and similar methods include the genetics of complex diseases and congenital defects, the identification of functional mutations in model organisms, and evolutionary genetics.     

Physical mapping of the rice genome with YAC clones

Construction of a rice physical map covered by YAC clones which have been arranged over half of the genome length is presented here. A total of 1285 RFLP and RAPD markers almost evenly distributed on the rice genetic map could select 2974 YAC clones and 2443 clones of them were located on their original positions. Rice YACs carrying 350 kb average insert fragments of 2443 clones could cover 222 megabase length of the rice genome, corresponding to 52% of the whole genome size (4.3 Mb). Chromosome landing with many YAC clones on the high-density genetic map loci efficiently integrated the genetic map with a physical map. This is the first step to generate a comprehensive genome map of rice. An integrated genome map should be an indispensable tool to figure out genome structure as well as to clone trait genes by map-based cloning.     

人类基因组中巢式基因对的系统分析

基因组上一个基因的所有或大部分外显子位于另一基因内含子和UTR中被称为巢式基因(Nested gene)。巢式基因对(Nested gene pair)是由主基因(Host gene)和巢式基因组成的基因对。文章针对人类基因组上的顺式巢式基因对(简称为巢式基因对)进行全基因组鉴定,并通过比较巢式基因对在人和小鼠基因组上的保守性分布,研究巢式基因对的进化方式。保守性分析表明基因转座、序列原位突变和主基因转录起始/终止位点突变是巢式基因对进化的主要原因,其中主基因转录起始/终止位点突变是巢式基因对独特的一种进化方式。Gene On-tology分析揭示大部分巢式基因与主基因的产物在功能上没有相关性。