Characterization of progenies of Triticum aestivum- Psathyrostachys juncea derivatives by using genomic in-situ hybridization

Using genomicin-situ hybridization (GISH) technique, 7 translocation-addition lines, 6 translocation and translocation-addition lines, 2 ditelosomic addition lines and 1 translocation line were identified fromTriticum aestivum L. -Psathyrostachys juncea (Fisch.) Nevski intergeneric hybrids, of which translocation-addition and translocation and translocation-addition lines were not found in other reports. No substitutions and disornic additions were detected in the, hybrids and breakages occurred in allP. juncea chromosomes studied. Results have shown that the improved GISH technique is a rapid and economical method for use in this field.     

Characterization of progenies of Triticum aestivum- Psathyrostachys juncea derivatives by using genomic in-situ hybridization

Using genomic in-situ hybridization (GISH) technique, 7 translocation-addition lines, 6 transloca-tion and translocation-addition lines, 2 ditelosomic addition lines and 1 translocation line were identified from Triticum aestivum L.-Psathyrostachys juncea (Fisch. ) Nevski intergeneric hybrids, of which translocation-addition and translocation and translocation-addition lines were not found in other reports. No substitutions and disomic additions were detected in the hybrids and breakages occurred in all P. juncea chromosomes studied. Results have shown that the improved GISH technique is a rapid and economical method for use in this field.     

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Comparative study ofTriticum aestivum andT. timopheevi genomes using C-banding technique

The somatic chromosomes ofTriticum timopheevi and those of two varieties ofT. aestivum, Chinese Spring and Bezostaya-1, have been identified by a Giemsa staining technique. The data suggest thatT. timopheevi and tetraploid wheats had a common ancestor from which their genomes differentiated due to chromosomal aberrations and the increase of heterochromatin in the chromosomes of theT. timopheevi G-genome. The differences between the chromosomes of the AB and AG genomes result in substitutions and large translocations between these chromosomes in interspecific hybrids.     

芥菜型多室油菜与甘蓝型油菜的种间远缘杂交

通过对芥菜型多室油菜与甘蓝型油菜种间杂交 以下简写为芥×甘或甘×芥 的结实性、交配性以及不同甘蓝型油菜对交配性的影响等研究发现:芥、甘正反交形成的饱满种子数较少,其形成种子的能力弱,但是芥×甘与甘×芥杂交相比,芥×甘形成饱满种子的能力较强,受精能力以及杂种胚胎的发育能力也强,在授粉后的子房发育上二者无显著差异.所以,芥菜型多室油菜与甘蓝型油菜种间杂交创建新资源时宜采用芥×甘杂交方式;不同甘蓝型油菜品种与芥菜型多室油菜正反交的结角率、受精指数、结籽指数和可交配指数均不相同,但可交配指数的变异系数最大.因此,筛选可交配性强的甘蓝型基因型应着眼于可交配指数高的甘蓝型油菜亲本材料,根据本试验结果,芥菜型多室油菜与甘蓝型油菜93-221-1杂交形成的杂种胚具有较强的可发育性.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

Interspecific hybridization between Brassica juncea and B. spinescens through protoplast fusion

Hypocotyl derived protoplasts of B. juncea cv. RLM-198 were fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids. Fusion products could be microscopically identified by characteristics of the protoplasts of both parents in the hybrid cells; they are colourless and vacuolated like the hypocotyl protoplasts and possess chloroplasts of the mesophyll protoplasts. The heterokaryotic fusion frequency was around 5%. However, the frequency of calli regenerating hybrid shoots was more than 10% of the regenerating calli. Putative somatic hybrids had morphological features characteristic of both the parents. Twelve plants analysed cytologically, possessed 52 chromosomes (26_II) at meiosis representing the complete genomes of B. juncea (18_II) and B. spinescens (8_II). For esterase isozymes, the hybrids had bands of Doth the parents. Hybrid nature of some of the plants was confirmed by their close resemblance to B. juncea, chromosome number and isozyme bands of B. spinescens as in Rsp-19. Somatic hybrids had rudimentary, non-dehiscent anthers and completely sterile pollen. However, on back crossing with B. juncea, 10 out of 12 plants produced seeds and about 100 plants were realized.Abbreviations PEG Polyethylene glycol     

Characterization of breast cancer by array comparative genomic hybridization.

Cancer progression is due to the accumulation of recurrent genomic alterations that induce growth advantage and clonal expansion. Most of these genomic changes can be detected using the array comparative genomic hybridization (CGH) technique. The accurate classification of these genomic alterations is expected to have an important impact on translational and basic research. Here we review recent advances in CGH technology used in the characterization of different features of breast cancer. First, we present bioinformatics methods that have been developed for the analysis of CGH arrays; next, we discuss the use of array CGH technology to classify tumor stages and to identify and stratify subgroups of patients with different prognoses and clinical behaviors. We finish our review with a discussion of how CGH arrays are being used to identify oncogenes, tumor suppressor genes, and breast cancer susceptibility genes.     

Characterization of mouse parvovirus infection by in situ hybridization.

Infection of young adult BALB/cByJ mice with mouse parvovirus-1, a newly recognized, lymphocytotropic, nonpathogenic parvovirus, was examined by in situ hybridization. Virus appeared to enter through the small intestine and was disseminated to the liver and lymphoid tissues. Strand-specific probes detected virion DNA in a consistently larger number of cells than replicative forms of viral DNA and/or viral mRNA. The number of signal-positive cells in the intestinal mucosa, lymph nodes, spleen, and thymus increased through day 10 after oral inoculation but decreased after seroconversion. Positive cells were still detected, however, in peripheral lymphoid tissues of mice examined at 9 weeks postinoculation. The results underscore the need to assess potential effects of persistent mouse parvovirus-1 infection on immune function in mice.     

Marker-based mapping of quantitative trait loci using replicated progenies

Summary When heritability of the trait under investigation is low, replicated progenies can bring about a major reduction in the number of individuals that need to be scored for marker genotype in determining linkage between marker loci and quantitative trait loci (QTL). Savings are greatest when heritability of the trait is low, but are much reduced when heritability of the quantitative trait is moderate to high. Required numbers for recombinant inbred lines will be greater than those required for a simple F₂ population when heritabilities are moderate to high and the proportion of recombination between marker locus and quantitative trait locus is substantial.Contribution No. 2613-E of the Agricultural Research Organization, 1989 series     

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

利用定量杂交方法对几个人工和环境微生物样品的分析研究

利用真细菌和真核生物两个域特异性探针,初步建立起16SrRNA定量杂交的方法,并对几个人工RNA样品（由提取自S.cerevisiae和P.fluorescens纯培养细胞的RNA按一定比例混合而成）和一个提取自垃圾填埋场渗滤液的RNA样品进行了初步的定量分析。结果表明该方法能从域的水平上对这些样品的组成作较精确的分析,从而在微生物生态学领域具有广泛的应用前景。     

Qualified testing of single-locus codominant inheritance using single tree progenies

In forest trees, classical techniques of studying modes of inheritance are usually not feasible due to the difficulty of performing controlled crosses. The limited information on inheritance extractable from readily available data, such as the large progenies collectable from single seed trees, must be compensated by the design of appropriately parameterized models. For this purpose, a system analytic approach is used to develop a new inferential framework for testing a single-locus codominant mode of inheritance of genetic traits using the inferred genotypes within progenies of single trees of inferred heterozygous genotype. Model assumptions are random gametic fusion between the local gamete pools and absence of postzygotic selection; ovule segregation distortion is allowed. The method yields estimates of the allele frequencies in both local gamete pools. Since tests of modes of inheritance must be tests of models rather than of parameters, the utility of the classical statistical testing procedures is limited, particularly concerning the qualification of a sampling method to attain a preassigned level of precision. Consistent application of this principle makes it possible to design qualified sampling methods prior to the actual experiment as well as to specify qualification levels for tests of completed experiments.     

DNA sequencing by hybridization using semi-degenerate bases.

One way to enhance the performance of hybridization microarrrays for DNA de novo sequencing is the use of probing patterns with gaps of unsampled positions. Ideally, such gaps could be realized by the inclusion into microarray oligos (probes) of wild-card compounds, referred to as universal bases (which bind nonspecifically to natural bases). The suggested alternative is to deploy in the gap positions degenerate bases, i.e., uniform mixtures of the four natural bases, with ensuing deterioration of the hybridization signal. In this paper, we show that such signal loss is a minor shortcoming, compared with the fact that degenerate bases cannot be treated as universal. Indeed, the substantial spread of hybridization energies at any microarray feature is such that on overwhelming number of mismatches bind more strongly than legal matches. We observed, however, that much narrower energy spreads are exhibited by pairs of bases in the same strength class (A-T and C-G). We call semi-degenerate a gap position realized with bases in the same energy class and show that well-known sequence reconstruction algorithms can be modified to achieve substantial improvements in sequencing effectiveness. For example, with a 4(9)-feature microarray and an acceptable weakening of the hybridization signal, one may achieve lengths of about 4,000 bases (compared with < 250 of the standard uniform method). Our approach also incorporates the use of a spectrum expressed in terms of observed feature melting temperatures (analog spectrum), rather than binary decisions made directly at the biochemical level (digital spectrum). While universal bases represent the ultimate goal of sequencing by hybridization, semidegenerate natural bases are the most effective known substitute.     

Characterization of double minute chromosomes’ DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH), and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. Received: 30 October 1994 / Revised: 25 February 1996     

Characterization of a de novo duplication of 11p14----p13, using fluorescent in situ hybridization and southern hybridization

A de novo 11p+ chromosome was found in a child with mild mental retardation but no other remarkable dysmorphic characteristics. Banding studies suggested a duplication of regions 11p13 and 11p14 or regions 11p14 and 11p15. Using fluorescent in situ hybridization and digital imaging microscopy, we mapped probe p32.1 (D11S16) to the proximal part of region 11p14 (11p14.1) and demonstrated duplication of this probe in our patient. Southern hybridization showed duplication of p32.1 and other probes located at 11p13 and 11p14, but the gene for alpha calcitonin (CALCA), located at 11p15, was not duplicated. The application of these techniques led to the identification of the duplication as dir dup(11)(pter----p13::p15.1----qter).     

Characterization of de novo duplications in eight patients by using fluorescence in situ hybridization with chromosome-specific DNA libraries.

Fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries was performed on samples from eight patients with de novo chromosomal duplications. In five cases, the clinical phenotype and/or cytogenetic evaluations suggested a likely origin of the duplicated material. In the remaining three cases, careful examination of the GTG-banding pattern indicated multiple possible origins; hybridization with more than one chromosome-specific library was performed on two of these cases. In all cases, FISH conclusively identified the chromosomal origin of the duplicated material. In addition, the hybridization pattern was useful in quantitatively delineating the duplication in two cases.