Chromosomal DNA Sequences Research Articles

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an ∼250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Human chromosome 7: DNA sequence and biology.

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.

New Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in Lactobacillus gasseri

Derivatives of a cryptic plasmid from Lactobacillus curvatus showed temperature-sensitive replication in thermophilic lactobacilli. The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences. pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35 degrees C, whereas replication is efficiently shut down at 42 degrees C. To demonstrate the functionality of pTN1, the signal transduction genes (nisRK) of the nisin-controlled expression system were integrated downstream of the pepN gene into the chromosome of Lactobacillus gasseri. In the resulting strain, UKLbg1, expression of nisRK was likely driven by cotranscription with pepN and enabled nisin-dependent induction of a fusion of a reporter gene (pepI) to the nisA promoter. The induction rates were correlated with the amount of nisin used, and maximum pepI expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited.

Abnormal tissue-dependent polytenization of a block of chromosome 3 pericentric heterochromatin in Drosophila melanogaster.

Heterochromatic DNA sequences in the polytene chromosomes of Drosophila melanogaster salivary glands are under-replicated in wild-type strains. In salivary glands of SuUR and in the nurse cells of otu mutants, under-replication is partly suppressed and a banded structure appears within the centric heterochromatin of chromosome 3. This novel banded structure in salivary gland chromosomes was called Plato Atlantis. In order to characterize the heterochromatic component of Plato Atlantis, we constructed a fine-scale cytogenetic map of deletions with break points within centric heterochromatin (Df(3L)1-16, Df(3L)2-66, Df(3R)10-65, Df(3R)4-75 and Df(3L)6B-29 + Df(3R)6B-29). Salivary gland chromosomes show that Df(3L)1-16 removes the complete Plato Atlantis, while Df(3L)2-66 deletes the most proximal 3L regions. These deletions therefore show a substantial cytological overlap. However, in the chromosomes of nurse cells, the same deficiencies remove distinct heterochromatic blocks, with the region of overlap being almost invisible. Satellite (AATAACATAG, AAGAG) and dodecasatellite DNAs mapped in a narrow interval in salivary glands but were found in three clearly distinguishable blocks in nurse cells. The 1.688 satellite was found at a single site in salivary glands but at two sites in nurse cells. We show that newly polytenized heterochromatic structures include blocks h47-h50d of mitotic heterochromatin in salivary glands, but the additional blocks h50p, h53 and h57 are also included in nurse cell chromosomes. Tissue specificity of the patterns of abnormal heterochromatic polytenization implies differential control of DNA replication in somatic and germline cells.

The DNA sequence and analysis of human chromosome 14.

Chromosome 14 is one of five acrocentric chromosomes in the human genome. These chromosomes are characterized by a heterochromatic short arm that contains essentially ribosomal RNA genes, and a euchromatic long arm in which most, if not all, of the protein-coding genes are located. The finished sequence of human chromosome 14 comprises 87,410,661 base pairs, representing 100% of its euchromatic portion, in a single continuous segment covering the entire long arm with no gaps. Two loci of crucial importance for the immune system, as well as more than 60 disease genes, have been localized so far on chromosome 14. We identified 1,050 genes and gene fragments, and 393 pseudogenes. On the basis of comparisons with other vertebrate genomes, we estimate that more than 96% of the chromosome 14 genes have been annotated. From an analysis of the CpG island occurrences, we estimate that 70% of these annotated genes are complete at their 5' end.

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.

Chromosomal fragility in patients with triple A syndrome.

Triple A syndrome is a rare, autosomal recessive disorder characterized by alacrima, achalasia, and adrenal insufficiency. Previous studies have shown that the triple A gene (AAAS) maps to chromosomal band 12q13. Mutations in the AAAS gene have been identified in triple A syndrome patients; however, the function of this gene is still obscure. We used classical and high-resolution chromosome analyses along with chromosome painting and DNA sequencing to study patients with triple A syndrome. We observed abnormalities in the heterochromatic region of chromosome 9 that included chromatid breaks, chromosome breaks, whole chromosome arm loss, and marker chromosomes, which occurred at unusually high frequencies in affected patients and heterozygotes. Our study raises the possibility of an association between chromosomal fragility in band 9q12 and triple A syndrome. Further investigation is necessary to understand the biologic basis of this finding in the context of triple A syndrome.

Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures.

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.

Characterization of regions of the cyanobacterial tRNA(pro) gene that affect the expression of a beta-glucuronidase reporter gene.

The E3 strong promoter-active fragment harbors the tRNA(pro) (GGG) gene upstream of the promoterless beta-glucuronidase (GUS) reporter gene in plasmid pKG-E3. The 74-bp tRNA(pro) coding sequence contains two regions exhibiting strong homology to blocks A and B which are the split promoter elements of eukaryotic tRNA genes. Results in this study showed that the promoter region of tRNA(pro) gene located upstream of its coding sequence and harbored the putative -10 (TACATT) and -35 (TTGGCA) regions which conformed to the Escherichia coli sigma(70) promoter. Differentiation of the 5' end of tRNA(pro)-GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions -3, -4, and -6, while the processed sites were located at position +75, +76 and +78 with respect to the first nucleotide of the tRNA(pro) coding sequence. The presence of block A decreased GUS activity about three-fold, whereas block B and the 3' end of tRNA(pro) gene completely abolished GUS expression. However, the presence of full-length tRNA(pro) gene did not affect the GUS expression. Downstream of the tRNA(pro) coding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted DeltaG value of -21.7 kcal x mol(-1). The absence of this stem-loop structure downstream of the tRNA(pro) coding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene.

Characterization of regions of the cyanobacterial tRNA pro gene that affect the expression of a β-glucuronidase reporter gene

The E3 strong promoter-active fragment harbors the tRNA pro (GGG) gene upstream of the promoterless β-glucuronidase ( GUS) reporter gene in plasmid pKG-E3. The 74-bp tRNA pro coding sequence contains two regions exhibiting strong homology to blocks A and B which are the split promoter elements of eukaryotic tRNA genes. Results in this study showed that the promoter region of tRNA pro gene located upstream of its coding sequence and harbored the putative −10 (TACATT) and −35 (TTGGCA) regions which conformed to the Escherichia coli σ 70 promoter. Differentiation of the 5′ end of tRNA pro- GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions −3, −4, and −6, while the processed sites were located at position +75, +76 and +78 with respect to the first nucleotide of the tRNA pro coding sequence. The presence of block A decreased GUS activity about three-fold, whereas block B and the 3′ end of tRNA pro gene completely abolished GUS expression. However, the presence of full-length tRNA pro gene did not affect the GUS expression. Downstream of the tRNA pro coding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted Δ G value of −21.7 kcal mol −1. The absence of this stem-loop structure downstream of the tRNA pro coding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene.

Mitotic stability of a coding DNA sequence-free version of Leishmania major chromosome 1 generated by targeted chromosome fragmentation

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a ‘mirror’ inverted duplication of the ‘right’ end of chromosome 1a (∼25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over ∼105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb ‘switch’ region) was found. By contrast, XC155 contains two large (∼13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp ‘satellite’ DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp ‘satellite’ DNA bound by telomeric repeats.

What we have learned from the DNA sequences of human chromosomes 21 and 22

What we have learned from the DNA sequences of human chromosomes 21 and 22

The DNA sequence and comparative analysis of human chromosome 20.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

The anatomy of the human genome: a neo-Vesalian basis for medicine in the 21st century.

Since 1956, the anatomy of the human genome has been described on the basis of chromosome studies, gene mapping, and DNA sequencing. The gross anatomy of Andreas Vesalius, published in 1543, played a leading role in the development of modern medicine. The objective of this article is to show that knowledge of genomic anatomy is having a comparably strong and pervasive influence on all of medicine. The research revealing human genome anatomy is reviewed. The insight provided by genome anatomy has brought about shifts of focus, both in research and in the clinic, eg, from genomics to proteomic and from the individually rare, single-gene disorders to common disorders. Genomic anatomy permits medicine to become more predictive and preventive. At the same time, diagnosis and treatment are rendered more sensitive, specific, effective, and safe. Hazards in misuse and misunderstanding of the information exist. Education of both the public and health professionals is vital if the full benefits of neo-Vesalian medicine are to be realized.

Statistical analysis of the DNA sequence of human chromosome 22.

We study statistical patterns in the DNA sequence of human chromosome 22, the first completely sequenced human chromosome. We find that (i). the 33.4 x 10(6) nucleotide long human chromosome exhibits long-range power-law correlations over more than four orders of magnitude, (ii). the entropies H(n) of the frequency distribution of oligonucleotides of length n (n-mers) grow sublinearly with increasing n, indicating the presence of higher-order correlations for all of the studied lengths 1<or=n<or=10, and (iii). the generalized entropies H(n)(q) of n-mers decrease monotonically with increasing q and the decay of H(n)(q) with q becomes steeper with increasing n<or=10, indicating that the frequency distribution of oligonucleotides becomes increasingly nonuniform as the length n increases. We investigate to what degree known biological features may explain the observed statistical patterns. We find that (iv). the presence of interspersed repeats may cause the sublinear increase of H(n) with n, and that (v). the presence of monomeric tandem repeats as well as the suppression of CG dinucleotides may cause the observed decay of H(n)(q) with q.

Murine models for Down syndrome

The availability of the recently published DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS). Trisomy 21 or DS is the only autosomal aneuploidy that is not lethal in the fetal or early postnatal period. DS phenotypes show variable penetrance, affecting many different organs, including brain (mental retardation, early onset of Alzheimer's disease, AD), muscle (hypotonia), skeleton, and blood. DS phenotypes may stem directly from the cumulative effect of overexpression of specific HSA21 gene products or indirectly through the interaction of these gene products with the whole genome, transcriptome, or proteome. Mouse genetic models have played an important role in the elucidation of the contribution of specific genes to the DS phenotype. To date, the strategies used for modeling DS in mice have been three: (1) to assess single-gene contributions to DS phenotype, using transgenic techniques to create models overexpressing single or combinations of genes, (2) to assess the effects of overexpressing large foreign DNA pieces, introduced on yeast artificial chromosomes (YACs) or bacterial artificial chromosomes (BACs) into transgenic mice, and (3) mouse trisomies that carry all or part of MMU16, which has regions of conserved homology with HSA21. Here we review the existing murine models and the relevance of their contribution to DS research.

GmhX, a novel gene required for the incorporation of L-glycero-D-manno-heptose into lipooligosaccharide in Neisseria meningitidis.

Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS (4.6 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of Escherichia coli HisB and to a family of genes of unknown function. The gene, designated gmhX, is part of a polycistronic operon (ice-2) containing two other genes, nlaB and orfC. nlaB encodes a lysophosphatidic-acid acyltransferase and orfC is predicted to encode a N-acetyltransferase. Specific polar and non-polar gmhX mutations in the parental strain, NMB, exhibited the truncated LOS structure of mutant 469, and repair of gmhX mutants by homologous recombination with the wild-type gmhX restored the LOS parental phenotype. GmhX mutants demonstrated increased sensitivity to polymyxin B. GmhX mutants and other Kdo(2)-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but were not as sensitive as inner-core mutants containing heptose. In the genomes of Helicobacter pylori and Synechocystis, gmhX homologues are associated with heptose biosynthesis genes; however, in N. meningitidis, gmhX was found in a location distinct from that of gmhA, rfaD, rfaE, aut and rfaC. GmhX is a novel enzyme required for the incorporation of L-glycero-D-manno-heptose into meningococcal LOS, and is a candidate for the 2-D-glycero-manno-heptose phosphatase of the heptose biosynthesis pathway.

DNA Mismatch Repair Enzyme Activity and Gene Expression in Prostate Cancer

Microsatellite instability (MSI) of short repetitive sequences in human chromosomal DNA can result from defective DNA mismatch repair function in tumor cells. We hypothesize that DNA mismatch repair (MMR) activity is down-regulated during prostatic carcinogenesis. To test this hypothesis, MMR activities and mismatch repair-related genes were analyzed in five different prostate cancer cell lines. Our results demonstrate that MMR activities were decreased as compared to MMR proficient HeLa cells. Interestingly, LNCaP, PC-3 and DU145 had much lower MMR activities as compared to DUPro and TSUPr1. The MMR-related genes (hMLH1, hPMS1, hPMS2, hMSH2, hMSH3, hMSH6) showed mRNA transcripts in all prostate cancer cell lines. However, Western blotting showed decreased or absent hMLH1 protein expression in PC-3, DU145, DUPro and TSUPr1 cells. Similarly, the hMSH2 protein expression was low or absent in DU145 and LNCaP cells. This is the first report that demonstrates decreased MMR activities is associated with low expression of hMLH1, hMSH2 and other MMR-related proteins in prostate cancer.

Organization of Human Cardiovascular-expressed Genes on Chromosomes 21 and 22

The recent availability of the sequenced and annotated DNA sequences of chromosomes 21 and 22 has initiated the next phase in the human genome project: the application of this resource. One facet of these data is that they provide a list of ordered genes along the chromosome that can be capitalized upon to determine gene position effects. Specifically, the physical position and distribution of genes along the chromosomes may be related to gene expression in specific organs or organ systems. In this report we index the subset of genes constituting the human “cardiovascular genome” on chromosomes 21q and 22q as well as report the identification of several “cardiovascular gene” clusters. These gene clusters are suggestive of a higher order of tissue-specific gene regulation at the chromosomal level.

Mutations in the NMMHC-A gene cause autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly/Sebastian syndrome)

AbstractMacrothrombocytopenia with leukocyte inclusions is a rare autosomal dominant platelet disorder characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. A previous study mapped a locus for the disease on chromosome 22q12.3-q13.2 by genome-wide linkage analysis. In addition, the complete DNA sequence of human chromosome 22 allowed a positional candidate approach, and results here indicate that the gene encoding nonmuscle myosin heavy chain-A, NMMHC-A, is mutated in this disorder. Mutations were found in 6 of 7 Japanese families studied: 3 missense mutations, a nonsense mutation, and a one-base deletion resulting in a premature termination. Immunofluorescence studies revealed that NMMHC-A distribution in neutrophils appeared to mimic the inclusion bodies. These results provide evidence for the involvement of abnormal NMMHC-A in the formation of leukocyte inclusions and also in platelet morphogenesis.