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PubMed1974

Dissection of chromosome structure with trypsin and nucleases.

Exhaustive digestion of chromatin with trypsin leads to the cleavage of only 20-30 amino acids from each of histones III (f3), IV (f2a1), IIb2 (f2b), and IIb1 (f2a1), the remainder of these chains being resistant. This resistance is not altered by removing the histones from the DNA with 2 M NaCl, but is dramatically reduced in 6 M urea. Histones III, IV, IIb2, and possibly IIb1 are cleaved at their N-termini. Histones I and V and the nonhistone proteins are the first to be attacked by trypsin and have no detectable trypsin-resistant fragments. The arginine rich histones, III and IV, are then cleaved as a pair, followed by most of IIb2 and IIb1, also as a pair. This data is consistent with a model in which basic N-terminal "arms" extend from a trypsin-resistant histone complex. The structural arrangement of these arms relative to the trypsin-resistant histone complex may define the spatial coordinates of DNA binding sites and, consequently, the folding of the DNA fiber in the chromosome. Accompanying the tryptic digestion of the N-terminals of histones III, IV, IIb2, and possibly IIb1, is an increased sensitivity of chromatin to staphylococcal nuclease. As analyzed by electrophoresis, untrypsinized chromatin is digested into eight discrete limit-digest fragments by nuclease. Trypsinization results in the nuclease digestion of some, but not all, of these DNA bands. Together with the information on the way trypsin cleaves histones in chromatin, the analysis of the resistant DNA suggests that histone N-terminals are associated with some DNA bands and histone C-terminals with other DNA bands. We propose that histones fold the chromosome by crosslinking the DNA corresponding to these bands.

Animals↗
PubMed1981

The chromosome structure and the cell cycle of Caulobacter crescentus. Isolation and analysis of envelope-free nucleoids.

Envelope-free nucleoids were isolated from an asymmetrically dividing bacterium, Caulobacter crescentus. In the nucleoid fraction, most of the DNA and nascent RNA in the cell and about 2% of the total cellular proteins were recovered. The sedimentation coefficient of the nucleoid was constant (1260 S) during the G1 period of the swarmer cell cycle and increased to 1940 S during the S period. Since both replicating (S period) and non-replicating (G1 period) chromosomes had a similar superhelical concentration, the increase in the sedimentation coefficient was simply explained by duplication of the nucleoid structure. The duplicated nucleoid was shown to segregate prior to the cell division. The pulse-labeled proteins recovered in the nucleoid fraction contained several stage-specific species, most of which were detected at the beginning of S period.

Bacterial Proteins↗
PubMed1997

Structure, chromosomal localization and expression of mouse genes encoding type III Reg, RegIII alpha, RegIII beta, RegIII gamma.

Reg (regenerating gene), first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet beta-cells. Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, Reg family, which consists of three subtypes (type I, II, III) based on the primary structures of the encoded proteins of the genes. In mouse, type I and type II Reg genes (i.e. RegI and RegII gene) have so far been isolated. In the present study, the complete nucleotide (nt) sequences of the cDNAs and genes encoding murine type III Reg (regenerating gene product), RegIII alpha, RegIII beta and RegIII gamma were determined. RegIII alpha, RegIII beta and RegIII gamma encode 175-, 175- and 174-amino acid (aa) proteins, respectively, with 60-70% homology. All three genes are composed of six exons and five introns spanning approx. 3 kb, and exhibit distinctive structural features unique for members of the Reg gene family. All the mouse Reg genes, RegIII alpha, RegIII beta, RegIII gamma, RegI and RegII, are assigned to the adjacent site of chromosome 6C by fluorescence in situ hybridization (FISH). RegIII alpha, RegIII beta and RegIII gamma were expressed weakly in pancreas, strongly in intestinal tract, but not in hyperplastic islets, whereas both RegI and RegII were expressed in hyperplastic islets. These results suggest that genes of the mouse Reg family are derived from a common ancestor gene by several gene duplications, and have obtained divergency in expression and function in the process of genetic evolution.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of the gene for a new epithelial beta-defensin. Genomic structure, chromosomal localization, and evidence for its constitutive expression.

Mammalian beta-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine beta-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans approximately 1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15. 1 within a region of conserved synteny to the chromosomal locations of human and mouse alpha- and beta-defensins. We also provide several independent lines of evidence showing that the pBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial beta-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-alpha, and interleukin (IL)-1beta failed to up-regulate the expression of pBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium or Actinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1beta. The constitutive expression of pBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-kappaB or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.

3T3 Cells↗
PubMed2001

Genomic structure, chromosome mapping and expression analysis of the human AXIN2 gene.

Conductin is a Wnt signalling protein and serves as a negative regulator of beta-catenin stability. We have previously isolated the human homolog (AXIN2) of the murine conductin gene and shown that it is mutated in colorectal cancer (CRC) with defective mismatch repair (MMR). Here we report the detailed genomic structure of this gene by analysis of cDNA and genomic clones. The gene spans > or =25 kb containing ten exons ranging from 96 bp to 904 bp. All splice donor and acceptor sites conform to the GT/AG rule. FISH (Fluorescence in situ Hybridization) analysis localized this gene to human chromosome band 17q24 and showed that it exists as a single copy in the human genome. Northern blot analysis from different human organs demonstrated that the AXIN2 gene is highly expressed in human thymus, prostate, testis, small intestine and ovarian tissues but expressed at a lower level in colon. The data reported here provides a framework for further analysis of this important Wnt signalling protein in vertebrate development and tumorigenesis.

Axin Protein↗
PubMed2001

Dual colour FISH in paraffin wax embedded bone trephines for identification of numerical and structural chromosomal abnormalities in acute myeloid leukaemia and myelodysplasia.

AIMS/BACKGROUND: The advent of new treatments for haematological malignancies has led to the need for a correlation between cytogenetic and morphological abnormalities. This study aimed to achieve this by the application of interphase cytogenetics to marrow trephine sections, a technique not previously reported for formalin fixed, paraffin wax embedded trephine biopsies. METHODS: Dual colour fluorescence in situ hybridisation (FISH) was used to detect numerical and structural abnormalities in routinely processed paraffin wax embedded trephine biopsies. Three cases with t(8;21) and three with t(15;17) were analysed, together with a case of trisomy 8. Chromosome specific probes were hybridised with sections and disclosed by fluorescein isothiocyanate and rhodamine/Texas red labelled antidigoxigenin and antibiotin amplification; translocations were identified by colocalisation of probes using a double wavelength bypass filter. RESULTS: A translocation signal was present in 12% and 11.5% of the cells counted in the t(8;21) and t(15;17) cases, respectively, but in none of the normal controls (p < 0.001). In the case of trisomy 8, 9% of the cells counted contained three hybridisation signals for chromosome 8, whereas no cell contained more than two in the normal control (p < 0.001). CONCLUSIONS: This technique is useful for archived routinely processed material, enabling it to be used as a research tool but also, and perhaps more importantly, in clinical practice.

Acute Disease↗
PubMed1999

Genomic structure, chromosomal mapping, and expression pattern of human DCAMKL1 (KIAA0369), a homologue of DCX (XLIS).

Human DCAMKL1, also known as KIAA0369, is a homologue of DCX (Xq22. 3), a gene associated with X-linked lissencephaly and subcortical band heterotopia. This suggests that DCAMKL1 may play a role in neuronal migration. The gene also shows similarity to Ca2+/calmodulin-dependent protein kinases. We have determined its genomic structure, regional mapping, and expression pattern in human tissues. DCAMKL1 consists of at least 18 exons ranging from 58 to 3359 bp in length. We have characterized the exon/intron borders, and primers were designed to amplify each individual exon for mutation analysis. DCAMKL1 was mapped to chromosome 13q13 by fluorescence in situ hybridization. Northern blot analysis showed DCAMKL1 to be predominantly expressed in human fetal brain as a major transcript of about 5.8 kb.

Brain↗
PubMed1996

The molecular structure, chromosomal organization, and interspecies distribution of a family of tandemly repeated DNA sequences of Antirrhinum majus L.

Monomers of a major family of tandemly repeated DNA sequences of Antirrhinum majus have been cloned and characterized. The repeats are 163-167 bp long, contain on average 60% A+T residues, and are organized in head-to-tail orientation. According to site-specific methylation differences two subsets of repeating units can be distinguished. Fluorescent in situ hybridization revealed that the repeats are localized at centromeric regions of six of the eight chromosome pairs of A. majus with substantial differences in array size. The monomeric unit shows no homologies to other plant satellite DNAs. The repeat exists in a similar copy number and conserved size in the genomes of six European species of the genus Antirrhinum. Tandemly repeated DNA sequences with homology to the cloned monomer were also found in the North American section Saerorhinum, indicating that this satellite DNA might be of ancient origin and was probably already present in the ancestral genome of both sections.

Base Sequence↗
PubMed1963

THE RELATION BETWEEN DNA SYNTHESIS AND CHROMOSOME STRUCTURE AS RESOLVED BY X-RAY DAMAGE.

Vicia faba root tip cells were treated for short periods with tritiated thymidine, either immediately before or after exposure of roots to x-rays, and autoradiograph preparations were analysed in an attempt to test the hypothesis that chromatid type (B') aberrations are induced only in those chromosome regions that have synthesized DNA prior to x-irradiation, whereas chromosome type (B'') aberrations are induced only in unduplicated chromosome regions. Studying the relation between presence or absence of label at loci involved in aberrations, in cells irradiated at different development stages, and the pattern of labelling in cells carrying both types of aberration leads to the conclusion that B'' aberrations are induced only in unreplicated chromosome regions. Following replication, only B' aberrations are induced, but these aberrations are also induced in chromosome regions preparing to incorporate DNA. It is suggested that the doubled response of the chromosome to x-rays prior to DNA incorporation might reflect a physical separation of replicating units prior to replication. The aberration yields in damaged cells which were irradiated in G(1)S, and early G(2) were in the ratio of 1.0:2.0:3.2. The data indicate that the increased yield of B' in early G(2) relative to S cells may be a consequence of changes in the spatial distribution of the chromosomes within the nucleus.

Chromatids↗
PubMed1994

Structure, chromosomal localization, and expression of 12 genes of the MAGE family.

We reported previously that human gene MAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with a MAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12 MAGE genes are located in the q terminal region of chromosome X. Like MAGE-1, the 11 additional MAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that of MAGE-1. The coding sequences of the MAGE genes predict the same main structural features for all MAGE proteins. In contrast, the promoters and first exons of the 12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of each MAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of the MAGE family including MAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Amino Acid Sequence↗
PubMedDate not supplied

[The effect of 5-azacytidine on the cell cycle and chromosome structure in cell cultures of swine embryonal kidney tissue].

Incubation of pig kidney cells (PK-cells) in the presence of 5-azacytidine (5-azaC), a DNA enzymatic methylation inhibitor, at concentration of 20 microM for 6 or 24 h results in a dramatic decrease in the DNA methylation level (5mC/C + m5.100) - from 3.0 in control to 1.0 in experiment. This is accompanied by a virtually complete arrest of mitosis and a decrease in the ratio of labeled interphase cells upon simultaneous introduction of 3H-deoxycytidine. The incubation with 5-azaC block PK-cells mainly in the G2-period. The inhibitory action is reversible, for the cells enter into mitosis after removal of the inhibitor. Metaphase chromosomes, whose DNA was replicated in the presence of the 5-azaC, exhibit certain ultrastructural differences from normal ones. The results are being discussed in connection with the earlier data on the anomalous structure of interphase chromatin formed in the course undermethylated DNA replication.

Animals↗
PubMed1994

Metaphase chromosome structure: bands arise from a differential folding path of the highly AT-rich scaffold.

Using the highly AT-specific fluorochrome daunomycin, a longitudinal optical signal called AT queue, thought to arise from a line-up of the highly AT-rich scaffold-associated regions (SARs) by the scaffolding, was identified in native chromosomes. Fluorescence banding is proposed to result from a differential folding path of the AT queue during its progression from telomere to telomere. The AT queue is tightly coiled or folded in a Q band, the resulting transverse striations across the chromatid, which also represent Giemsa subbands, generating a bright AT-rich signal over the Q region. The R bands, in contrast, contain a more central (unfolded) AT queue, yielding an AT-dull signal over the R regions. The AT queue is identified by immunofluorescence against topoisomerase II (topo II) and HMG-I/Y as the scaffold of native chromosomes; the fluorescence signal from both proteins is akin to a detailed Q-type banding pattern. Native chromosomes appear assembled according to the loop-scaffold model.

Animals↗