Distribution and sequence analysis of the centromere-associated repetitive element CEN38 of Sorghum bicolor (Poaceae)

Fluorescence in situ hybridization (FISH) of a large-insert genomic clone, BAC 22B2, previously suggested that Sorghum bicolor (2n = 20) has the tetraploid architecture A(b)A(b)B(b)B(b). Here, we report on BAC 22B2 subclone pCEN38 (1047-bp insert) as related to sorghum and sugarcane. Mitotic FISH of six different subclones of BAC 22B2 showed that pCEN38 produced the strongest specificity to the A(b) subgenome and signal occurred primarily near centromeres. Southern blots of pCEN38 to 21 crop plants revealed a narrow taxonomic distribution. Meiotic metaphase I FISH positioned pCEN38 sequences near active centromeres. Pachytene FISH revealed that the distributions are trimodal in several B(b) and possibly all sorghum chromosomes. DNA sequencing revealed that the pCEN38 fragment contains three tandemly repeated dimers (<280 bp) of the same sequence family found in sorghum clone pSau3A10, and that each dimer consists of two divergent monomers (<140 bp). Sequence comparisons revealed homology between the pCEN38 monomers and the SCEN 140 bp tandem repeat family of sugarcane. FISH of pCEN38 yielded signal in centromere regions of most but not all sugarcane chromosomes. Results suggest that sugarcane and sorghum share at least one ancestor harboring elements similar to pCEN38 and SCEN and that each species had an ancestor in which the repetitive element was weakly present or lacking.     

Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.     

Identification and characterisation of sugarcane intergeneric hybrids,Saccharum officinarum x Erianthus arundinaceus,with molecular markers and DNA in situ hybridisation

Molecular markers were used to characterise sugarcane intergeneric hybrids between S. officinarum and E. arundinaceus. Very simple diagnostic tools for hybrid identification among the progeny were derived from isozyme electrophoresis and a sequence-tagged PCR. Two enzyme systems (GOT and MDH B) and PCR amplification revealing spacer-size variation in the 5s-rDNA cluster were found most convenient. Specific characterisation of the two genomic components was possible using RFLP and in situ hybridisation. The strong molecular differentiation between S. officinarum and E. arundinaceus allows the identification of numerous Erianthus-specific RFLP bands in the hybrids. Genomic DNA in situ hybridisation allows for the differentiation of the chromosomes contributed by S. officinarum and E. arundinaceus in chromosome preparations of the hybrids. In situ hybridisation with the 18s-5.8s-25s rDNA probe highlights the basic chromosome numbers in the two parental species. The potential of these techniques to monitor the Erianthus genome during the introgression process is discussed.     

Postsynaptic density antigens: preparation and characterization of an antiserum against postsynaptic densities

Long-term immunization of rabbits with postsynaptic densities (PSD) from bovine brain produced an antiserum specific for PSD as judged by binding to subcellular fractions and immunohistochemical location at the light and electron microscope levels. (a) The major antigens of bovine PSD preparations were three polypeptides of molecular weight 95,000 (PSD-95), 82,000 (PSD-82), and 72,000 (PSD-72), respectively. Antigen PSD-95, also present in mouse and rat PSDs was virtually absent from cytoplasm, myelin, mitochondria, and microsomes from rodent or bovine brain. Antigens PSD-82 and PSD-72 were present in all subcellular fractions from bovine brain, especially in mitochondria, but were almost absent from rodent brain. The antiserum also contained low-affinity antibodies against tubulin. (b)Immunohistochemical studies were performed in mouse and rat brain, where antigen PSD-95 accounted for 90 percent of the antiserum binding after adsorption with purified brain tubulin. At the light microscope level, antibody binding was observed only in those regions of the brain where synapses are known to be present. No reaction was observed in myelinated tracts, in the neuronal cytoplasm, or in nonneuronal cells. Strong reactivity was observed in the molecular layer of the dentate gyrus, stratum oriens and stratum radiatum of the hippocampus, and the molecular layer of the cerebellum. Experimental lesions, such as ablation of the rat entorhinal cortex or intraventricular injection of kainic acid, which led to a major loss of PSD in well- defined areas of the hippocampal formation, caused a correlative decrease in immunoreactivity in these areas. Abnormal patterns of immunohistochemical staining correlated with abnormal synaptic patterns in the cerebella of reeler and staggerer mouse mutants. (c) At the electron microscopic level, immunoreactivity was detectable only in PSD. The antibody did not bind to myelin, mitochondria or plasma membranes. (d) The results indicate that antigen PSD-95 is located predominantly or exclusively in PSD and can be used as a marker during subcellular fractionation. Other potential uses include the study of synaptogenesis, and the detection of changes in synapse number after experimental perturbations of the nervous system.     

Evolution of interspersed repetitive elements inGossypium (Malvaceae)

Very little is known regarding how repetitive elements evolve inpolyploid organisms. Here we address this subject by fluorescent insitu hybridization (FISH) of 20 interspersed repetitive elements tometaphase chromosomes of the cotton AD-genome tetraploid Gossypiumhirsutum and its putative A- and D-genome diploid ancestors. Theseelements collectively represent an estimated 18% of the G.hirsutum genome, and constitute the majority of high-copyinterspersed repetitive elements in G. hirsutum. Seventeen ofthe elements yielded FISH signals on chromosomes of both G.hirsutum subgenomes, while three were A-subgenome specific. Hybridization of eight selected elements, two of which were A-subgenomespecific, to the A(2) genome of G. arboreum yielded asignal distribution that was similar to that of the G. hirsutumA-subgenome. However, when hybridized to the D(5) genome ofG. raimondii, the putative diploid ancestor of the G.hirsutum D-subgenome, none of the probes, including elements thatstrongly hybridized to both G. hirsutum subgenomes, yieldeddetectable signal. The results suggest that the majority, although notall, G. hirsutum interspersed repetitive elements haveundergone intergenomic concerted evolution following polyploidizationand that this has involved colonization of the D-subgenome byA-subgenome elements and/or replacement of D-subgenome elements byelements of the A-subgenometype.     

FISH of a maize sh2-selected sorghum BAC to chromosomes of Sorghum bicolor.

Fluorescence in situ hybridization (FISH) of a 205 kb Sorghum bicolor bacterial artificial chromosome (BAC) containing a sequence complementary to maize sh2 cDNA produced a large pair of FISH signals at one end of a midsize metacentric chromosome of S. bicolor. Three pairs of signals were observed in metaphase spreads of chromosomes of a sorghum plant containing an extra copy of one arm of the sorghum chromosome arbitrarily designated with the letter D. Therefore, the sequence cloned in this BAC must reside in the arm of chromosome D represented by this monotelosome. This demonstrates a novel procedure for physically mapping cloned genes or other single-copy sequences by FISH, sh2 in this case, by using BACs containing their complementary sequences. The results reported herein suggest homology, at least in part, between one arm of chromosome D in sorghum and the long arm of chromosome 3 in maize.     

ISH-facilitated analysis of meiotic bivalent pairing.

Chiasmata constitute one of the cornerstones of sexual reproduction in most eukaryotes. They mediate the reciprocal genetic exchange between homologues and are essential to the proper orientation of the homologous centromeres in meiosis I. As markers of recombination, they offer a cytological means of mapping. Rather than trying to accurately count individual chiasmata, we have examined properties of the mathematical relationship between frequencies of nonadorned disomic configurations in meiosis (ring, rods, and univalents) and the probabilities at which arms of the respective chromosomes are chiasmate (one or more chiasma per arm). Numerical analyses indicated that conventionally analyzed bivalents with nonidentified arms yield statistically biased estimates of chiasma probabilities under a broad range of circumstances. We subsequently analyzed estimators derived from adorned configurations with ISH-marked arms, which were found to be statistically far superior, and with no assumptions concerning interference across the centromere. We applied this methodology in the study of chromosomes 16 and 23 of cotton (Gossypium hirsutum), and estimated their arm lengths in centimorgans. The results for chromosome 23, the only one of the two chromosomes with a documented RFLP map, were consistent with the literature. Similar molecular-meiotic configuration analyses can be used for a wide variety of eukaryotic organisms and purposes: for example, providing far more powerful meiotic comparisons of genomes of chromosomes, and a rapid means of evaluating effects on recombination. Key words : meiotic configurations, chiasma frequencies, in situ hybridization, cotton.     

Bioassay-guided discovery of antibacterial agents: in vitro screening of Peperomia vulcanica, Peperomia fernandopoioana and Scleria striatinux

Background

The global burden of bacterial infections is high and has been further aggravated by increasing resistance to antibiotics. In the search for novel antibacterials, three medicinal plants: Peperomia vulcanica, Peperomia fernandopoioana (Piperaceae) and Scleria striatinux (Cyperaceae), were investigated for antibacterial activity and toxicity.

Methods

Crude extracts of these plants were tested by the disc diffusion method against six bacterial test organisms followed by bio-assay guided fractionation, isolation and testing of pure compounds. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were measured by the microdilution method. The acute toxicity of the active extracts and cytotoxicity of the active compound were performed in mice and mammalian cells, respectively.

Results

The diameter of the zones of inhibition (DZI) of the extracts ranged from 7?C13?mm on Escherichia coli and Staphylococcus aureus of which the methylene chloride:methanol [1:1] extract of Scleria striatinux recorded the highest activity (DZI?=?13?mm). Twenty-nine pure compounds were screened and one, Okundoperoxide, isolated from S. striatinux, recorded a DZI ranging from 10?C19?mm on S. aureus. The MICs and MBCs indicated that the Peperomias had broad-spectrum bacteriostatic activity. Toxicity tests showed that Okundoperoxide may have a low risk of toxicity with an LC₅₀ of 46.88???g/mL.

Conclusions

The antibacterial activity of these plants supports their use in traditional medicine. The pure compound, Okundoperoxide, may yield new antibacterial lead compounds following medicinal chemistry exploration.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.