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B A Hamkalo

Publications and source records attributed to B A Hamkalo.

At least 19 recordsLinked to original sources
PubMed1992

Cytological and molecular characterization of centromeres in Mus domesticus and Mus spretus.

We have applied EM in situ hybridization (EMISH) and pulsed field gel electrophoresis (PFGE) to samples from diploid primary cell cultures and an established cell line to examine in detail the relative organization of the major and minor satellite DNAs and telomere sequences in the genomes of Mus domesticus and Mus spretus. EMISH localizes the Mus domesticus minor satellite to a single site at the centromere-proximal end of each chromosome. Double label hybridizations with both minor satellite and telomere probes show that they are in close proximity and possibly are linked. In fact, PFGE of M. domesticus DNA digested with Sal I and Sfi I reveals the presence of fragments which hybridize to both probes and is consistent with the physical linkage of these two sequences. The M. domesticus minor satellite is the more abundant satellite in Mus spretus. Its distribution in M. spretus is characterized by diffuse labeling with no obvious concentration near chromosome ends. In addition to this repeat the M. spretus genome contains a small amount of DNA that hybridizes to a M. domesticus major satellite probe. Unlike the M. domesticus minor satellite, it is not telomere proximal but is confined to a domain at the border of the centromere and the long arm. Thus, although both species possess all three sequences, except for the telomeres, their distribution relative to one another is not conserved. Based on the results presented, we propose preliminary molecular maps of the centromere regions of Mus domesticus and Mus spretus.

Animals↗
PubMed1992

Hoechst 33258, distamycin A, and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA.

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.

Animals↗
PubMed1991

Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli.

The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope. The haploid yeast genome contains 100-200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure. We combined in situ hybridization of non-isotopically labeled probes to isolated S. cerevisiae nuclei with immunogold detection to localize rDNA and rDNA precursor sequences in nuclei at the electron microscope (EM) level. Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus. In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus. These data, in conjunction with published immunofluorescent localizations of nucleolar-associated antigens, provide definitive proof that the dense crescent is the nucleolus. Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.

Base Sequence↗
PubMed1991

DNA sequence mapping using electron microscopy.

DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.

Acetoxyacetylaminofluorene↗
PubMed1991

Preferential distribution of active RNA polymerase II molecules in the nuclear periphery.

We have combined immunogold labeling with the Miller spreading technique in order to localize proteins at the electron microscope (EM) level in whole mount nuclei from mouse and human fibroblasts. Anti-histone H1 antibody labels nuclei uniformly, indicating that the nuclear interior is accessible to both antibodies and gold conjugates. Anti-topoisomerase I antibody labels nucleoli intensely, in agreement with previous immunofluorescent and biochemical data. Two different antibodies against the large subunit of RNA polymerase II (pol II) show preferential labeling of the nuclear periphery, as do antibodies against lamin, a known peripheral nuclear protein. Treatment of cells with alpha-amanitin results in loss of virtually all RNA polymerase II staining, supporting the specificity of labeling. Finally, when nuclei are incubated in the presence of biotin-UTP (bio-UTP) under run-off transcription conditions, incorporation is preferentially located at the nuclear periphery. These results support the conclusions that transcriptionally active pol II molecules are non-uniformly distributed in fibroblast nuclei, and that their differential distribution mirrors that of total pol II.

Amanitins↗
PubMed1990

High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization.

We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.

Animals↗
PubMed1989

Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.

Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions necessary to perform multiple sequence localization and signal amplification.

Animals↗
PubMed1987

Curvature of mouse satellite DNA and condensation of heterochromatin.

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.

Animals↗
PubMed1986

Chinese hamster cells with a minichromosome containing the centromere region of human chromosome 1.

We describe a series of primary and secondary hamster-human hybrids which have selectively retained a small amount of human DNA. The hybrid XJM12.1.3 contains an estimated 4000-8000 kb of human DNA, and for a secondary hybrid derived from it, XEW8.2.3, our estimate is 1000-2000 kb. The hybridization of Southern blots of DNA from these hybrids with a variety of human satellite DNA probes reveals that these lines include centromere sequences of human chromosome 1. The identifiable human DNA is in the form of a minichromosome, as detected by in situ hybridization in the light microscope and in the electron microscope. At mitosis, the minichromosome can be observed to have kinetochores and to be associated with microtubules. Therefore, it can segregate in a stable fashion. It may be significant that in the selection of the hybrids we had selected for a human gene which has been mapped on human chromosome 1.

Animals↗
PubMed1986

Early replication and expression of oocyte-type 5S RNA genes in a Xenopus somatic cell line carrying a translocation.

In Xenopus somatic cells, the somatic-type 5S RNA genes replicate early in S phase, bind the transcription factor TFIIIA, and are expressed; in contrast, the late replicating oocyte-type genes do not bind TFIIIA and are transcriptionally inactive. These facts support a model in which the order of replication of the somatic-type versus the oocyte-type 5S genes causes their differential expression in somatic cells due to sequestration of TFIIIA by the early-replicating somatic genes. Here we provide further evidence for the model by showing that in one Xenopus cell line in which some oocyte-type 5S genes are translocated, some oocyte-type 5S genes replicate early and are expressed.

Animals↗
PubMed1986

Mouse satellite DNA, centromere structure, and sister chromatid pairing.

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

Animals↗
PubMed1985

Ultrastructural features of minute chromosomes in a methotrexate-resistant mouse 3T3 cell line.

The Miller spreading procedure was applied to mouse metaphase spreads of methotrexate-resistant 3T3 cells that contain large numbers of minute chromosomes and dihydrofolate reductase genes. There is substantial variation in both size and numbers of minutes in individual cells, the smallest of which (estimated as 5 X 10(3) kilobase pairs) would be undetected by standard light microscopic analyses. Minute chromosomes are composed of nucleosomal chromatin, which is organized into typical higher order fibers that are folded to form rosette-like structures characteristic of normal chromosome organization. There is no evidence that the DNA in minutes is linear. Minutes exist singly and in pairs, and members of a pair are connected by higher order chromatin fibers, suggesting that they are topologically interlocked. They are often closely apposed to chromosomal telomeres or arms, a configuration that may be involved in their distribution at mitosis. In addition to typical minutes, which do not possess kinetochores, a small marker chromosome possessing all of the features of a centromere region is present in parental and resistant cells. An unusual feature of this cell line is the retention of resistance, minute chromosomes, and amplified dihydrofolate reductase genes; most methotrexate-resistant mouse cell lines with minute chromosomes lose these properties when grown in the absence of methotrexate.

Animals↗
PubMed1979

Adrenocortical cytochrome P-450 side chain cleavage. Preparation of membrane-bound side chain cleavage system from purified components.

Soluble cytochrome P-450 from bovine adrenocortical mitochrondria, capable of side chain cleavage, can be incorporated into membranes prepared by dispersion of phospholipids in aqueous buffer when cholate is added to the membrane suspension. In addition, the complete protein side chain cleavae system (i.e. including the ancillary proteins adrenodoxin and adrenodoxin reductase and the substrate cholesterol) can be incorporated into such membranes so that on addition of TPNH, pregnenolone is formed. These components remain in the membrane through gel filtration (which removes almost all the cholate) and sedimentation through sucrose density gradients which separate vesicles without protein and soluble enzyme from the membrane-bound P-450 remains associated with the membrane during and following lysis of vesicles. The vesicles which do not leak [14C]glucose were seen on electron microscopy to show a mean diameter of 350 to 450 A. A number of phospholipids are capable of accomodating P-450 in this manner: mitochondrial lipid extracts, synthetic dipalmitoyl phosphatidylcholine, synthetic dipalmitoyl phosphatidylserine, and egg lecithin, separately or in various combinations. Cholesterol is not necessary for incorporation of the side chain cleavage system. Membrane-bound P-450 shows a Vmax of 28.1 nmol of pregnenolone/min/mg of protein, more than 10 times that of soluble P-450. The spectral properties of the soluble P-450 are altered to become predominantly low spin in the membrane and the enzyme is more stable at 4 degrees C than is soluble p-450.

Adrenal Cortex↗
PubMed1979

Nucleosome packing in interphase chromatin.

Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.

Animals↗
PubMed1978

Higher order structure in metaphase chromosomes. I. The 250 A fiber.

Metaphase chromosomes released from cells in the presence of Joklik's suspension media by vortex-mixing with 0.5 mm glass beads have been analyzed by electron microscopy. In these preparations the chromosomes are composed of series of loops (200-300 A in diameter) which are, in turn, composed of closely-apposed arrays of nucleosomes. Negative-staining of these preparations has allowed the identification of several distinct patterns within the loop which appear to arise from variations in nucleosome packing. Analogous patterns are also observed in chromatin fragments generated by brief micrococcal nuclease digestion. From these data we have deduced certain features of nucleosome-nucleosome interactions in higher-ordered chromatin fibers.

Animals↗
PubMed1978

Higher order structure in metaphase chromosomes. II. The relationship between the 250 A fiber, superbeads and beads-on-a-string.

The morphology of metaphase chromosome-derived chromatin fibers released from cells by non-ionic detergent cell lysis in the presence of divalent cations has been studied by electron microscopy. In these preparations the euchromatic arms appear as a series of loops, 200-300 A in diameter, which are composed of closely-apposed nucleosome arrays. The higher order fiber in chromosomes derived from detergent-lysed cells appears to be less stable than chromatin fibers obtained by mechanical cell lysis. The fiber breaks down into a series of non-uniform nucleosome aggregates (superbeads) and finally to chromatin in a beads-on-a-string morphology upon incubation at 31 degrees for 20 min. These observations allow us to suggest a relationship between uniform thick fibers, superbead-containing fibers, and beads-on-a-string chromatin within metaphase chromosomes.

Animals↗
PubMed1978

Selective digestion of mouse metaphase chromosomes.

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000 X g pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.

Animals↗