Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy

A fluorescent in situ hybridization procedure with a chromosome 1-specific (1q12) repetitive satellite DNA probe was used to label the 1q12 regions of the chromosomes 1 in spherical and polymorphic hemopoietic cell nuclei. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The 1q12 regions of chromosome 1 were measured to be closely associated with the nuclear envelope in isolated nuclei of unstimulated diploid human lymphocytes. The relative positions to each other in the periphery of these spherical nuclei could not be distinguished from a random distribution pattern. In the diploid and tetraploid polymorphic nuclei of cells of the promyelocytic leukemia cell line HL60 these pericentromeric sequences were also associated with the nuclear surface.     

Increased level of prolactin gene sequences in bromodeoxyuridine treated GH cells.

The 5-bromodeoxyuridine-resistant (BrdUrdr) derivative (F1BGH12C1) of prolactin nonproducing (PRL-) rat pituitary tumor cell-subclone GH12C1, synthesize prolactin (PRL) in the presence of the drug. Analysis of nuclear RNA isolated from BrdUrd treated F1BHG12C1 cells demonstrated several high molecular weight RNA PRL sequences, similar to those observed in the nuclear RNA fraction of PRL producing (PRL+) GH3 cells. No such RNAPRL sequences could be detected in nuclear RNA fraction of untreated F1 BGH12C1 cells. PRL sequences in the genome of GH3 (PRL+), GH12C1 (PRL-) and F1BGH12C1 (PRL-, BrdUrdr) GH cells could be identified by blot analysis in 4.8-5.2kb fragment of restriction endonuclease, Hind III digested DNA. Both PRL+ and PRL- cells seem to have approximately the same level of PRL gene sequences in total cell DNA. However Hind III digested DNA of BrdUrd treated F1BGH12C cells revealed the presence of significantly higher levels of PRL gene sequences, in comparison, to that observed in total DNA of untreated cells. The increased level of PRL gene sequences was dependent on the period of drug treatment and a parallel increase in the cytoplasmic RNAPRL sequences was also observed.     

Broad range DNA probes for detecting and amplifying eubacterial nucleic acids

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.     

The genes for cytoplasmic ribosomal ribonucleic Acid in higher plants

The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 × 10⁶ molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction.     

Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.     

Chromosomal localization of chicken sequences homologous to the beta-actin gene by in situ hybridization

In situ DNA/chromosome hybridization techniques were used to localize the cytoplasmic beta-actin gene in the chicken. Hybridization of a beta-actin cDNA probe to metaphase chromosome spreads indicated that sequences complementary to this probe are located on the long arm of chromosome 2 (2q) and one of chromosomes 9 through 12.     

Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization.

Subregional localization of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12 has been performed using the fluorescence in situ hybridization (FISH) technique. The following order is suggested for the 13 mapped genes: cen-->COL2A1-->(VDR-D12S15)-->(D12S17-D12S4++ +-D12S14-D12S6)-->D12S8-->(IAPP-MGF- D12S7-D12S12)-->IGF1-->qter. Eight of the mapped genes clustered at two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 (IAPP-MGF-D12S7-D12S12). Our results show that single-copy DNA sequences as small as 500 bp can be successfully mapped by FISH.     

Typing of anastomosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA

A method based on restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA was developed for the rapid characterization of large populations of Rhizoctonia solani at the anastomosis group (AG) level. The restriction maps of the internal transcribed spacers (ITS) sequences were compared for 219 isolates of R. solani belonging to AG-1 to AG-12 and AG-BI, representing diverse geographic and host range origins. Four discriminant restriction enzymes (MseI, AvaII, HincII, and MunI) resolved 40 restriction fragment length polymorphism (RFLP) types among the 219 ITS sequences of R. solani. Each RFLP type could be assigned to a single AG except for two RFLP types, which were common to two AG. A fifth enzyme allowed the discrimination of AG-6 and AG-12. In addition, the combination of four enzymes allowed the discrimination of subsets within AG-1, AG-2, AG-3, and AG-4. The efficiency of the typing method was confirmed by analyzing PCR-amplified ITS sequences of 30 reference strains. Furthermore, the PCR-RFLP method was used to characterize at the AG level 307 isolates of R. solani originating from ten sugar beet fields exhibiting patches of diseased plants in France. The PCR-based procedure described in this paper provides a rapid method for AG typing in R. solani.     

Localization of enterobacterial common antigen immunoreactivity in the ribosomal cytoplasm of Escherichia coli cells cryosubstituted and embedded at low temperature.

The application of two on-section immunogold labeling techniques, the Lowicryl K4M (progressive lowering of temperature) procedure and the cryosection technique of Tokuyasu, in a previous work to study the topology of enterobacterial common antigen (ECA) biosynthesis revealed the presence of label on the outer membrane and in areas associated with the inner side of the cytoplasmic membrane. However, labeling was also observed in the ribosomal cytoplasm. The question of whether the cytoplasmic label was a result of ECA displacement during the more slowly acting aldehyde fixation or whether cytoplasmic ECA precursors are true constituents of the ribosomal cytoplasm could not be resolved from these results. In the study described here, cells of the same Escherichia coli F470 strain were reinvestigated by comparison of the progressive lowering of temperature and improved cryosubstitution-low-temperature embedment techniques. The latter procedure, applied directly to nonpretreated and noncentrifuged cells, led to superior ultrastructural preservation of the cytoplasmic organization, with little opportunity for cytoplasmic antigen displacement after the primary cryofixation step; the label distribution obtained supports the conclusion that N-acetylmannosaminuronic acid (ManNAcA)-containing ECA precursors are real constituents of the ribosomal cytoplasm. Results from tunicamycin inhibition studies of ECA biogenesis in the E. coli mutant 2465 suggested that even the ECA precursor UDP-ManNAcA alone or a chemically unidentified product(s) generated from accumulated ManNAcA residues may react with the monoclonal antibody used, leading to weak but clearly positive cytoplasmic labeling. The relatively intense labeling obtained with cells grown in the absence of the drug can be explained by the reactivity of further ManNAcA-containing ECA precursors with the monoclonal antibody used.     

New insights into the evolution of chromosome 1

A complex low-repetitive human DNA probe (BAC RP11-35B4) together with two microdissection-derived region-specific probes of the multicolor banding (MCB) probe-set for chromosome 1 were used to re-analyze the evolution of human chromosome 1 in comparison to four ape species. BAC RP11-35B4 derives from 1q21 and contains 143 kb of non-repetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12 and 1p36.1 of Homo sapiens (HSA). Human chromosome 1 was studied in comparison to its homologues in Hylobates lar (HLA), Pongo pygmaeus (PPY), Gorilla gorilla (GGO) and Pan troglodytes (PTR). A duplication of sequences homologous to human 1p36.1 could be detected in PPY plus an additional signal on PPY 16q. The region homologous to HSA 1p36.1 is also duplicated in HLA, and split onto chromosomes 7q and 9p; the region homologous to HSA 1q21/1p12 is present as one region on 5q. Additionally, the breakpoint of a small pericentric inversion in the evolution of human chromosome 1 compared to other great ape species could be refined. In summary, the results obtained here are in concordance with previous reports; however, there is evidence for a deletion of regions homologous to human 1p34.2-->p34.1 during evolution in the Pongidae branch after separation of PPY.     

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

用人染色体14q24.3区带探针池直接分离表达顺序

本文报道了从显微切割的人染色体区带直接分离区带专一性表达序列的方法和结果。     

The Male Sterility-Associated Pcf Gene and the Normal Atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Reassociation and dissociation of cytoplasmic membrane-associated DNA

The relationship between nuclear DNA and cytoplasmic membrane-associated DNA, extracted from a human lymphocyte cell line, was examined by DNA-DNA reannealing and by dissociation of renatured molecules. Up to 2% of the total cellular DNA is found in the cytoplasm as cytoplasmic membrane-associated DNA and of this 2%, approximately 70% is comprised of repeated sequences. These sequences are homologous to only about 4% of the repeated sequences of nuclear DNA. The repeat fraction of cytoplasmic membrane-associated DNA consists of sequences which are only moderately repeated. The number of copies in the average “family” could range from about 1500 copies to as few as 25 copies. A small rapidly reannealing portion of cytoplasmic membrane-associated DNA (C₀t < 4 × 10^?3) appears to consist of sequences derived from a single “family”.About 30% of cytoplasmic membrane-associated DNA reassociates slowly with a $\text{C}{}_0\text{t}\text{1}\text{2}$ value of 223 (unique cytoplasmic membrane-associated DNA). This fraction has homology with about 11% of the unique sequences of nuclear DNA. However, unique cytoplasmic membrane-associated DNA comprises only about 0·6% of the total cellular DNA. If it is assumed that each cell has the same amount of cytoplasmic membrane-associated DNA, homology with 11% of the unique sequences of nuclear DNA suggests that different cells may have different unique nucleotide sequences in the cytoplasm.     

Evolution and phylogenetic information content of the ITS-1 region in the tiger beetle Cicindela dorsalis

Sequence divergence in the internal transcribed spacer region 1 (ITS-1) of the ribosomal DNA locus was assessed in subspecies of the coastal North American tiger beetle, Cicindela dorsalis. The spacer region was amplified using the polymerase chain reaction and cloned for sequencing. Of a total of 50 clones obtained from 12 specimens, 42 clones were different in at least one nucleotide position. In a parsimony analysis of these sequences, the main phylogenetic distinction was found to separate sequences from the Gulf of Mexico and the Atlantic Ocean. Within these two assemblages phylogenetic resolution was low, and the variation within individuals was almost as high as the variation within the entire lineage. The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes. Polymorphisms of limited geographical distribution could be detected, and 41 additional clones were partly sequenced, to assess the geographic distribution of these polymorphisms in more detail. In a population aggregation analysis, the geographic pattern of ITS-1 distribution was basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.      

Two Classes of Cytoplasmic Viral RNA Synthesized Early in Productive Infection with Adenovirus 2

The RNA sequences and RNA size classes transcribed early in productive infection with adenovirus 2 were analyzed by RNA-DNA hybridization. Two independent procedures demonstrated that early cytoplasmic viral RNA is composed of two sequence classes, class I which is absent or present in greatly reduced quantities at 18 h, and class II which persists throughout the infection. When the sequences in early viral RNA were analyzed by hybridization-inhibition studies, the hybridization of early [(3)H]RNA was inhibited only 50% by RNA from cultures harvested late (18 h) in infection. Liquid hybridizations with radioactive viral DNA confirmed that early RNA includes two classes. Duplex formation of RNA with (32)P-labeled viral DNA was assayed by hydroxylapatite chromatography and resistance to S(1) nuclease digestion. Both methods showed that the cytoplasmic RNA present early in infection annealed 12 to 15% of the viral DNA; late cytoplasmic RNA hybridized 21 to 25% of the DNA. Mixtures of early plus late cytoplasmic RNAs hybridized 30 to 34% of the viral DNA, demonstrating the reduced concentration of early class I RNA in the late RNA preparations. Experiments were performed to correlate class I and class II early RNA with size-fractionated cytoplasmic RNA synthesized early in infection. Fractionation of RNA by gel electrophoresis or sucrose gradient centrifugation confirmed three major size classes, 12 to 15S, 19 to 20S, and 26S. Total cytoplasmic RNA and RNA selected on the basis of poly(A) content contained the same size classes of viral RNA. In standard electrophoresis conditions, the 19 to 20S viral RNA could be resolved into two size classes, and the distribution of 12 to 15S RNA also indicated the presence of more than one size component. Hybridization-inhibition studies under nonsaturating conditions were performed with 26S, 19 to 20S, and 12 to 15S viral RNAs fractionated by gel electrophoresis. Late RNA inhibited the hybridization of 26S RNA only 20%, 19 to 20S RNA was inhibited 45%, and 12 to 15S RNA was inhibited 50%. When 18 to 19S and 12 to 15S viral RNAs purified by sucrose gradient centrifugation were similarly analyzed, late RNA inhibited hybridization of 18 to 19S RNA 50%, and the annealing of 12 to 15S RNA was inhibited 70%.