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PubMed2004

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus.

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.

Archaeal Proteins↗
PubMed1996

Retinal-specific guanylate cyclase gene mutations in Leber's congenital amaurosis.

Leber's congenital amaurosis (LCA, MIM 204,000), the earliest and most severe form of inherited retinopathy, accounts for at least 5% of all inherited retinal dystrophies. This autosomal recessive condition is usually recognized at birth or during the first months of life in an infant with total blindness or greatly impaired vision, normal fundus and extinguished electroretinogram (ERG). Nystagmus (pendular type) and characteristic eye poking are frequently observed in the first months of life (digito-ocular sign of Franceschetti). Hypermetropia and keratoconus frequently develop in the course of the disease. The observation by Waardenburg of normal children born to affected parents supports the genetic heterogeneity of LCA. Until now, however, little was known about the pathophysiology of the disease, but LCA is usually regarded as the consequence of either impaired development of photoreceptors or extremely early degeneration of cells that have developed normally. We have recently mapped a gene for LCA to chromosome 17p13.1 (LCA1) by homozygosity mapping in consanguineous families of North African origin and provided evidence of genetic heterogeneity in our sample, as LCA1 accounted for 8/15 LCA families in our series. Here, we report two missense mutations (F589S) and two frameshift mutations (nt 460 del C, nt 693 del C) of the retinal guanylate cyclase (RETGC, GDB symbol GUC2D) gene in four unrelated LCA1 probands of North African ancestry and ascribe LCA1 to an impaired production of cGMP in the retina, with permanent closure of cGMP-gated cation channels.

Blindness↗
PubMed1986

Identification of 200,000-dalton human cell surface protein encoded by gene mapped to long arm of chromosome 11.

Cell surface proteins and glycoproteins of human and Chinese hamster cells and their hybrid cell clones were analyzed by two-dimensional gel electrophoresis. The J1 clone of human-Chinese hamster hybrid cells contained chromosome 11 as its only human chromosome. The J1 cells expressed a glycoprotein of 200,000 daltons which was shared by human fibroblasts but not by the parental Chinese hamster ovary cells. The 200,000-dalton protein was identified as a cell surface protein by the method of lactoperoxidase-catalyzed iodination. The protein was electrophoretically purified from radioiodinated cultures of human fibroblasts and J1 cells and subjected to the analysis of tryptic peptides by thin-layer electrophoresis followed by chromatography. The protein from both sources gave rise to fingerprints which closely resembled to each other. The results are consistent with a hypothesis that the 200,000-dalton protein of the J1 clone is of human origin. Analysis of segregant clones of J1 cells, which have deletions on human chromosome 11, has further suggested that the gene for this glycoprotein maps to the long arm of chromosome 11. A gene coding for the 200,000-dalton protein has not been previously mapped to this chromosome.

Animals↗
PubMed1990

[Isolation and mapping of arbitrary single copy DNA fragment located on human chromosome 11p11-q11].

A library of genomic DNA has been constructed in EMBL3 lambda phage, from a Chinese hamster/human lymphocytes somatic cell hybrid carrying human chromosome 11 and 20. Recombinants containing human genomic DNA origin can be isolated from the hybrid cell genomic library by using species-specific probe. 8 single copy fragments have been isolated from 13 recombinants. One of them designated as FD11-1 has been identified on chromosome 11 by hybridized it with hybrid cell clone panel and mapped on chromosome 11p11-q11 by chromosome in situ hybridization. On chromosome 11, 3 linkage groups were reported, which located on 11p15, 11p13 and 11q13 respectively. Therefore, the FD11-1 will supply a new locus on chromosome 11 for linkage analysis. Endonuclease recognizing sites and potential recognizing sites on FD11-1 will guide the further RFLP studies.

Chromosome Mapping↗
PubMed1998

Biochemical imaging and 2D classification of keratin pearl structures in oral squamous cell carcinoma.

Precise regulation of cell organization results in well-differentiated tissue structures and continuous renewal of the oral epithelium maintaining a highly ordered tissue architecture. Here we demonstrate that FT-IR microspectroscopy, performed on sections of cancerous tissue biopsies, is capable of generating biochemical maps that show the distribution and any abnormal concentration of individual classes of biomolecules. Oral epithelia affected by cancer (squamous cell carcinoma) show many abnormal changes in morphology, of which the formation of keratin pearls is only one. Spectra from selected pearl areas demonstrate that these structures contain not only abnormal keratin concentrations but also seem to be stabilized by surrounding collagen fibers. Infrared image maps reveal that in the center of keratin pearls the concentration of protein (cytokeratins) is abnormally high, that DNA is absent and that the cell membrane fluidity is reduced. This suggests that cells are structurally destroyed and transformed into nuclei-free horny cells, simulating normal differentiation and epithelial growth. We also introduce a new analysis modality, two-dimensional (2D) tissue classification, and apply it to establish spectral similarities between different tissue structures. A total of 315 spectra, recorded for the original map, were analyzed by pattern recognition methods, classified and re-assembled into new maps based on their spectral similarities. The re-assembled maps clearly indicate significant tissue changes outside the pearls, suggesting early biochemical changes that accompany abnormal growth. Employing this 2D analysis modality in combination with infrared histopathology may be relevant to tumor diagnosis and prognosis.

Cluster Analysis↗
PubMed1985

Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information.

The epithelial HBL-100 cell line was established in vitro from milk of an apparently healthy woman. It exhibits characteristics of transformation from the very beginning and evolves during in vitro maintenance, until becoming tumorigenic in nude mice. This immortal cell line represents a useful model for studying the progression of human epithelial cells toward malignancy. In the course of our investigations we detected a 94K protein in HBL-100 cells obtained from four different sources. This protein is shown to be indistinguishable from the SV40 large T-antigen on the basis of: Recognition by polyclonal and different monoclonal antibodies. Partial peptide map analysis. Specific binding capacity to the SV40 DNA origin of replication. The presence of a tandemly integrated SV40 genome is demonstrated by Southern blotting. Successful rescue of SV40 DNA by fusion with permissive COS-7, but not CV-1 cells, indicates that the SV40 T-antigen from HBL-100 cells is defective in a function(s) essential to the replication of the viral DNA. The possible origin of the SV40 genetic information that we have detected in HBL-100 cells and the implications of this finding on studies involving this cell line are discussed.

Adenocarcinoma↗
PubMed1975

Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants.

The envB1 mutation mediating a distorted cell morphology of Escherichia coliK-12 was cotransducible with strA, aroE, aspB, and argG. The mapping data is consistent with a gene location for envB around 62.5 min. In partial diploids envB1 was recessive to its wild-type allele. The original envB mutant contained a second mutation in a locus denoted sloB close to strA. The following gene order is suggested: sloB-strA-aroE-envB-aspB-argG. The sloB1 mutation caused a marked reduction in the growth rate of both envB and envB+ strains. Moreover, this mutation in the presence of envB1 appears to increase the ratio between deoxyribonucleic acid and protein in cells growing in rich medium. The phenotypic properties of envB1, sloB+ and envB+ transductants were characterized. Cells with envB1, sloB+ genotype were hypersensitive to several penicillins including the beta-lactam compound, amidino penicillin. Penicillin hypersensitivity could not be explained by increased outer membrane penetrability. The original envB mutant (envB1,SLOB1), as well as envB1, sloB1 or envB+, SLOB1 transductants were resistant to amidino penicillin. Resistance was explained by the slow growth rate mediated by the sloB1 mutation. The similarity between envB cells and wild-type cells treated with sublethal concentrations of amidino penicillin was emphasized.

Bacterial Proteins↗
PubMed1997

Target- as well as source-derived factors direct the morphogenesis of anomalous retino-thalamic projections.

Neonatal tectal lesions in hamsters result in the elimination of a major central target of retinal axons, massively denervate the lateral posterior nucleus of the thalamus (LP), and lead to a marked increase of the retino-LP projection. In such animals, retino-LP axons show all of the normally-occurring terminal types. In addition, large clusters of varicosities, whose tubular configuration resembles the major type of tecto-LP terminals observed in normal animals, are also noted if the tectal lesion is made on the day after birth (P1). If, however, the neonatal lesion occurs on P5 rather than on P1, terminals resembling normal tecto-LP endings are rarely observed; rather, the distribution and morphology of retino-LP terminals bear a greater resemblance to those seen in normal hamsters, but the size and complexity of the terminals, particularly those that form string-like arrangements, is significantly increased. Our findings suggest that the altered morphology of some abnormally induced retino-LP terminals may be orchestrated by target-associated signals. However, there are age-related limitations on the degree to which afferent systems can vary their terminal morphology; these restrictions may derive from the target, or may be a function of intrinsic changes within the cells of origin of the afferent fibers.

Animals↗
PubMed2000

Towards a two-dimensional proteome map of Mycoplasma pneumoniae.

A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.

Acrylic Resins↗
PubMed1992

Use of chimeras to study gene function in mesodermal tissues during gastrulation and early organogenesis.

The origin of different mesodermal tissues during gastrulation and the developmental lability of mesodermal precursors can be mapped by transplanting marked epiblast cells to the same or a different position in a host egg cylinder, and assessing the subsequent fate of transplanted tissue. This information provides the context for assessing the role of particular patterns of gene expression during mesoderm formation and differentiation. For example, the stability of Hox gene expression can be examined by transplanting transgenically marked somites that express a particular Hox gene to a position in the somite file where it is not normally expressed. Such experiments can reveal not only the cues required for Hox gene expression but also the relevance of a circumscribed pattern of Hox gene expression to a specific developmental fate. A different approach to resolving gene function is to mix mutant cells known to affect mesoderm formation with normal cells and to determine the cell autonomy of mutant cells in a normal environment. Homozygous Brachyury (T/T) embryonic stem cell lines have been isolated and injected into normal blastocysts. The presence of T/T cells in chimeras results in mesodermal defects similar to those seen in the intact mutant.

Animals↗
PubMed1996

Ectopic secretion of osteocalcin, the major non-collagenous bone protein, by the myeloma cell line NCI-H929.

In this study we provide the first evidence that human non-osteoblastic cells secrete the bone-specific protein osteocalcin. We show that the myeloma cell line NCI-H929 constitutively produces low amounts of osteocalcin. Furthermore, we demonstrate that this production is strongly enhanced after 1,25(OH)2D3 stimulation. An amplification or a deregulation of the osteocalcin gene could be responsible for the observed phenomenon because NCI-H929 has the unusual characteristic of having at least three chromosomes 1, where the osteocalcin gene was mapped (1q25-q31). It is of interest to note that the patient in whom the NCI-H929 cell line originated never developed osteolytic lesions despite extensive disease apparent at autopsy. Although lytic bone lesions with low osteocalcin serum levels are a common feature of multiple myeloma (MM), a small subset of patients does not develop lytic bone lesions or even demonstrate osteosclerotic MM. This occurrence is associated with high osteocalcin serum levels of unknown origin. In this context, osteocalcin production by immortalized tumor lacking any osteolytic potential could be relevant to the pathophysiology of this particular type of MM.

Blotting, Western↗
PubMed2002

DNase I sensitive site in the core region of the human beta-globin origin of replication.

HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin. DNA was isolated from the residual chromatin attached to the nuclear matrix, digested with Hind III, and subjected to hybridization with [(32)P] labeled probe located upstream of the core region of the human beta-globin replication origin. The hybridization pattern revealed the existence of a DNase I sensitive site in the core region of the beta-globin replicator. The results suggest that association with the nuclear matrix induce alteration in the chromatin structure of the origin of replication that represents a more open chromatin configuration.

Binding Sites↗
PubMed1986

Nonrandom chromosome structural aberrations and oncogene loci in human malignant melanoma.

Short-term cultures of 10 malignant melanomas derived from 8 patients were analyzed cytogenetically. The chromosome composition of the tumors was found to be similar in terms of modal number and structural and numerical aberrations, especially the nonrandom nature of breakpoints. Six chromosomes were consistently involved in marker formation. Aberrations of chromosomes #1 and #9 were identified in every tumor, whereas structural alterations of chromosome #2 were found in 9 tumors. In contrast, aberrations of chromosomes #6, #3, and #7 were identified in 7, 7, and 8 of the tumors, respectively. The nonrandom breakpoints on these chromosomes frequently coincided with known oncogenic loci and resulted in morphologically identical marker chromosomes. Consecutive lesions were obtained for two patients. Common markers were identified in both cases, indicating the clonal origin of the tumors. In addition, many marker chromosomes characteristic of the individual lesions were also identified. The presence of these lesion-specific markers indicates the nonrandom selective nature of the metastatic process and suggests the possible heterogeneity of the original tumor cell population.

Adult↗
PubMed1996

Origin of thalamocortical projections to the presupplementary motor area (pre-SMA) in the macaque monkey.

The presupplementary motor area (pre-SMA) is a recently defined cortical motor area that is located immediately rostral to the supplementary motor area (SMA) and is considered to play more complex roles in motor control than the SMA. In the present study, we examined the distribution of cells of origin of thalamocortical projections to the pre-SMA in the macaque monkey. Under the guidance of intracortical microstimulation mapping, the retrograde tracer biotinylated dextran amine was injected into the pre-SMA. Retrogradely labeled neurons were distributed primarily in the parvicellular division of the ventroanterior nucleus (VApc), oral division of the ventrolateral nucleus (VLo), area X, and mediodorsal nucleus (MD). Some labeled neurons were also observed in the medial and caudal divisions of the ventrolateral nucleus. The results indicate that the pre-SMA may receive not only basal ganglia inputs via the VApc, VLo, and MD, but also a cerebellar input via the X.

Animals↗
PubMed2001

The hippocampal debate: are we asking the right questions?

For years, the debate has been: "Is the hippocampus the cognitive map?" or "Is the hippocampus the core of memory?" These two hypotheses derived their original power from two key experiments--the cognitive map theory from the remarkable spatial correlates seen in recordings of hippocampal pyramidal cells and the memory theory from the profound amnesias seen in the patient H.M. Both of these key experiments have been reinterpreted over the years: hippocampal cells are correlated with much more than place and H.M. is missing much more than just his hippocampus. However, both theories are still debated today. The hippocampus clearly plays a role in both navigation and memory processing. The question that must be addressed is rather: "What is the role played by the hippocampus in the navigation and memory systems?" By looking at the navigation system as a whole, one can identify the major role played by the hippocampus as correcting for accumulation errors that occur within idiothetic navigation systems. This is most clearly experimentally evident as reorientation when an animal is lost. Carrying this over to a more general process, this becomes a role of recalling a context, bridging a contextual gap, or, in other words, it becomes a form of recognition memory. I will review recent experimental data which seems to support this theory over the more general spatial or memory theories traditionally applied to hippocampus.

Animals↗
PubMed1995

Chromatid and chromosome type breakage-fusion-bridge cycles in wheat (Triticum aestivum L.).

During the development of disomic additions of rye (Secale cereale L.) chromosomes to wheat (Triticum aestivum L.), two reverse tandem duplications on wheat chromosomes 3D and 4A were isolated. By virtue of their meiotic pairing, the reverse tandem duplications initiated the chromatid type of the breakage-fusion-bridge (BFB) cycle. This BFB cycle continued through pollen mitoses and in the early endosperm divisions, but no clear evidence of its presence in embryo mitoses was found. The chromosome type of BFB cycle was initiated by fusion of two broken chromosome ends resulting in a dicentric or a ring chromosome. Chromosome type BFB cycles were detected in embryo mitoses and in root tips, but they did not persist until the next meiosis and were not transmitted to the progeny. Active BFB cycles induced breakage of other wheat chromosomes that resulted in additional reverse tandem duplications and dicentric and ring chromosomes. Four loci, on chromosome arms 2BS, 3DS, 4AL, and most likely on 7DL, were particularly susceptible to breakage. The BFB cycles produced high frequency of variegation for pigmentation of the aleurone layer of kernels and somatic chimeras for a morphological marker. With the exception of low mutation rate, the observed phenomena are consistent with the activity of a Ds-like element. However, it is not clear whether such an element, if indeed present, was of wheat or rye origin.

Bread↗
PubMed1997

Replication initiation and elongation fork rates within a differentially expressed human multicopy locus in early S phase.

Replication of the 400 copies of the 43 kb human ribosomal RNA (rDNA) locus spans most of the S phase. To examine the basis for the unusual pattern of rDNA replication, a sensitive strategy was developed to map origins of DNA replication and measure apparent rates of fork progression within a chromosomal locus. This technique, termed differential intragenomic replication timing, revealed that initiation within the actively transcribed rDNA occurred in early S within a 10.7 kb region spanning the promoter and 5' external transcribed spacer. Forks emanating from this early bidirectional origin progressed at an apparent slow rate with the sense and anti-sense forks moving at 0.32 and 0.23 kb/min. Using a photochemical-based technique, the chromatin status of the rDNA repeats was assayed throughout the S phase. Approximately 85% of the rDNA repeats were in a transcriptionally active chromatin structure at the start of S phase. A progressive decrease in the transcription state of the rDNA loci was observed, reaching a minimum between 3 and 6 h in mid S phase. Altogether, the data suggest a link between RNA polymerase I mediated transcription and site-specific initiation of DNA replication within the rDNA multicopy locus.

Chromatin↗