Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.     

Resistance to antimycin A in yeast by amplification of ADH4 on a linear, 42 kb palindromic plasmid

A yeast strain lacking a functional copy of ADH1 has been isolated that is resistant to antimycin A because of the presence of multiple copies of a nuclear gene, ADH4. The amplified copies of ADH4 exist on linear molecules 42 kb in length, which can be separated from chromosomal DNA by orthogonal-field-alternation gel electrophoresis. These amplified molecules are palindromes that reanneal rapidly after denaturation to form linear, snap-back molecules 21 kb in length. The amplified ADH4 sequences are bounded by telomere-homologous sequences. The chromosomal copy of ADH4 is the most distal marker on the left arm of chromosome VII, and the amplified ADH4-containing molecules appear to contain two copies of the region extending from ADH4 to the telomere.     

Characterization of a mouse interferon gene locus I. Isolation of a cluster of four alpha interferon genes.

A BALB/c mouse genomic library was screened with a murine interferon alpha 2 (MuIFN-alpha 2) cDNA coding region fragment. Eight clones were isolated which contain different mouse chromosomal segments related to the MuIFN-alpha 2 probe and a 28 kilobase (kb) region of mouse genomic DNA containing four different MuIFN-alpha genes (alpha 1, alpha 4, alpha 5 and alpha 6) was identified and characterized; an intergenic 1000 nucleotide long conserved sequence was found to be associated with three of these four alpha genes, indicating that this alpha-IFN gene cluster evolved through tandem duplications. Sequence analysis revealed the absence of a polyadenylation site in the 3' untranslated region of MuIFN-alpha 1, and showed that one of the genes (alpha 4) contains an internal deletion of 5 amino acids in the coding region.     

Structure and chromosomal localization of the mouse oncomodulin gene

The rat gene encoding oncomodulin (OM), a small calcium-binding protein, is under the control of a solo LTR derived from an endogenous intracisternal A-particle. The latter sequence is the only OM promoter analyzed so far. In order to study cell-type-specific OM expression in a species lacking LTR sequences in the OM locus, we initially synthesized an OM cDNA from mouse placenta. By sequencing, we found a 137-bp-long 5 leader region that differed markedly from its rat counterpart but had high similarity to several mouse genomic sequences. Primers specific to this sequence in addition with primers specific for an exon 2/intron 2 sequence were used to screen a mouse ES cell line genomic P1 library. One positive clone contained the whole OM gene, including intron 1 of 25 kb and a 5 flanking region of 27 kb lacking an LTR. The region upstream of exon 1 contains no TATA or CCAAT boxes but has a homopurine/homopyrimidine stretch of 102 bp as well as a (CA)₂₂ repeat. The latter sequence is polymorphic and was therefore, used to map the OM gene to the distal end of the long arm of mouse Chromosome (Chr) 5 by interspecific backcross analysis. Additonally we localized the OM gene by in situ hybridization to the region G1-3 on Chr 5, confirming the genetic linkage results. Finally, the OM gene was found to be structurally conserved and to exist in a single copy in mammals.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

A modification of reverse chromosome painting was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16–24 copies; the largest unit was bigger than 600 kb and was present in 16–32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13–q15. Notably, the signal on 12q13–q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13–q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13–q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.     

Sequence analysis of a total of three megabases of DNA in two regions of chromosome 8p.

Large-scale sequencing of genomic regions and in silico gene trapping together represent a highly efficient and powerful approach for identifying novel genes. We performed megabase-level sequence analyses of two genomic regions on human chromosome 8p (8p11.2 and 8p22-->p21.3), after covering those segments with sequence-ready contigs composed of 74 cosmids, 14 BACs, and three PAC clones. We determined continuous nucleotide sequences of 1,856,753 bases on 8p11.2 and 1,210,381 bases on 8p22-->p21.3 by combining the shotgun and primer-walking methods. In silico gene trapping identified four novel genes in the 8p11.2 region and, in the 8p22-->p21.3 region, six known genes (PRLTS, PCM1, MTAMR7, HCAT2, HFREP-1 and PHP) and three novel genes. The distribution of Alu and LINE1 repetitive elements and the densities of predicted exons were different in each region, and Alu-rich portions contained more exonic sequences than LINE1-rich areas.     

Regional localization of th human EGF-like growth factor CRIPTO gene (TDGF-1) to chromosome 3p21

The CRIPTO gene encodes a novel human growth factor structurally related to epidermal growth factor. We localized the CRIPTO gene to chromosome 3p21 by fluorescence in situ hybridization with a cosmid clone containing 40 kb of the CRIPTO genomic region (TDGF-1). To suppress hybridization to CRIPTO-related sequences, present in multiple copies in the human genome, hybridization was carried out in the presence of unlabeled CRIPTO cDNA in excess over the probe. Our finding confirms the provisional mapping of the CRIPTO gene to chromosome 3, and assigns it precisely to a chromosomal region involved in several rearrangements occurring in malignancy.CRIPTO-specific sequences are present in multiple copies in the human genome (Dono et al. 1991). Two genomic CRIPTO-encoding sequences, TDGF-1 and TDGF-3, have been isolated and characterized. TDGF-1 corresponds to the structural gene encoding the protein expressed in teratocarcinoma cells (Ciccodicola et al. 1989). TDGF-3, possibly a functional pseudogene, corresponds to a complete copy of the TDGF-1 mRNA that contains seven base changes representing both silent and replacement substitutions in the coding region (Dono et al. 1991). By somatic cell hybrid analysis TDGF-1 has been assigned to chromosome 3, and TDGF-3 to the Xq21–22 region (Dono et al. 1991).     

Rice mitochondrial genome contains a rearranged chloroplast gene cluster

Summary We have previously reported the isolation and partial sequence analysis of a rice mitochondrial DNA fragment (6.9 kb) which contains a transferred copy of a chloroplast gene cluster coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), and subunits of ATPase (atpB and atpE), methionine tRNA (trnM) and valine tRNA (trnV). We have now completely sequenced this 6.9 kb fragment and found it to also contain a sequence homologous to the chloroplast gene coding for the ribosomal protein L2 (rpl2), beginning at a site 430 bp downstream from the termination codon of rbcL. In the chloroplast genome, two copies of rpl2 are located at distances of 20 kb and 40 kb, respectively, from rbcL. We have sequenced these two copies of rice chloroplast rpl2 and found their sequences to be identical. In addition, a 151 bp sequence located upstream of the chloroplast rpl2 coding region is also found in the 3 noncoding region of chloroplast rbcL and other as yet undefined locations in the rice chloroplast genome. Hybridization analysis revealed that this 151 bp repeat sequence identified in rice is also present in several copies in 11 other plant species we have examined. Findings from these studies suggest that the translocation of rpl2 to the rbcL gene cluster found in the rice mitochondrial genome might have occurred through homologous recombination between the 151 bp repeat sequence present in both rpl2 and rbcL.     

Characterization of six new loci within the swine major histocompatibility complex class III region

A search for new potential coding sequences was conducted within two overlapping cosmid genomic DNA clusters of about 170 and 45 kb from the swine major histocompatibility complex class III region. The sequences were detected with various probes, including pools of swine cDNA, homologous and heterologous genomic sequences, and synthetic oligonucleotides. The 170 kb cluster was centered on the tumor necrosis factor genes (TNF), and the 45 kb cluster contained the heat-shock protein 70 genes (HSP70). The TNF cluster revealed the presence of five new genes: lymphotoxin , BAT1, BAT2, BAT3, and a sequence related to DNA-binding factors. No sequence homologous to B144 was found in the TNF cluster, although other unidentified coding sequences may be present in this cluster. The HSP70 cluster contained a gene identified as BAT6, that is, tRNA-valyl synthetase. These results provide new evidence that the genomic maps of these various genes in the TNF and HSP70 sub-regions are similar in swine and human.     

Comparative sequence analysis of 634 kb of the mouse chromosome 16 region of conserved synteny with the human velocardiofacial syndrome region on chromosome 22q11.2

Mouse genomic DNA sequence extending 634 kb on proximal mouse chromosome 16 was compared to the corresponding human sequence from chromosome 22q11.2. Haploinsufficiency for this region results in velocardiofacial syndrome (VCFS) in humans. The mouse region is rearranged into three conserved blocks relative to human, but gene content and position are highly conserved within these blocks. Examination of the boundaries of one of these blocks suggested that the evolutionary chromosomal rearrangement occurred in the mouse lineage, resulting in inactivation of the mouse orthologue of ZNF74. Sequence analysis identified 21 genes and 15 ESTs. These include 2 novel genes, Srec2 and Cals2, and previously undescribed splice variants of several other genes. Exon discovery was carried out using GRAIL2, MZEF, or comparative analysis across 491 kb of conserved mouse and human sequence. Sequence comparison was highly effective, identifying every gene and nearly every exon without the high frequency of false-positive predictions seen when algorithmic methods were used alone. In combination, these procedures identified every gene with no false-positive predictions. Comparative sequence analysis also revealed regions of extensive conservation among noncoding sequences, accounting for 6% of the sequence. A library of such sequences has been established to form a resource for generalized studies of regulatory and structural elements.     

Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the ³H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.