Oat-maize chromosome addition lines: a new system for mapping the maize genome

Novel plants with individual maize chromosomes added to a complete oat genome have been recovered via embryo rescue from oat (Avena sativa L., 2n = 6x = 42) x maize (Zea mays L., 2n = 20) crosses. An oat-maize disomic addition line possessing 21 pairs of oat chromosomes and one maize chromosome 9 pair was used to construct a cosmid library. A multiprobe (mixture of labeled fragments used as a probe) of highly repetitive maize-specific sequences was used to selectively isolate cosmid clones containing maize genomic DNA. Hybridization of individual maize cosmid clones or their subcloned fragments to maize and oat genomic DNA revealed that most high, middle, or low copy number DNA sequences are maize-specific. Such DNA markers allow the identification of maize genomic DNA in an oat genomic background. Chimeric cosmid clones were not found; apparently, significant exchanges of genetic material had not occurred between the maize-addition chromosome and the oat genome in these novel plants or in the cloning process. About 95% of clones selected at random from a maize genomic cosmid library could be detected by the multiprobe. The ability to selectively detect maize sequences in an oat background enables us to consider oat as a host for the cloning of specific maize chromosomes or maize chromosome segments. Introgressing maize chromosome segments into the oat genome via irradiation should allow the construction of a library of overlapping fragments for each maize chromosome to be used for developing a physical map of the maize genome.     

Psychopathology, treatment completion and 5 years outcome. A prospective study of drug abusers

The strategy of isolating the band-specific expression fragments from a probe pool generated by human chromosome microdissection was reported. A chromosome 14q24.3 band-specific single copy DNA pool was constructed based on this probe pool. Using total DNA of the pool as probe to hybridize the human marrow cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Among these primary clones, 32 secondary clones were obtained after second-round screening and designed as cFD14-1-32. Finally, 24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization. Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but can't hybridize to 17q11-12 DNA. Partial sequences of 13 fragments of them were sequenced and identified as novel cDNA sequences, and these sequences were proved to have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.     

Metastasizing placental site trophoblastic tumor: immunohistochemical and DNA analysis. 2 case reports and a review of the literature

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.     

The possibility of using highly disperse iron for the directed transport of thyroxin in the body

In order to isolate promoters of mouse TGF-beta receptor genes, we used inverse PCR with highly overlapped primers corresponding to the 5' sequence of the receptor cDNAs. Nested primer sets only covered a 30- to 40-base region of the sequences. HinfI-digested and self-ligated mouse genomic DNA was used as a PCR template. Only one band for each receptor was seen after PCR. The amplified DNA fragments could direct luciferase production when the luciferase coding sequence was ligated after the fragments. The sequence of the fragment which correspond to the type II receptor showed partial homology with the promoter region of the human TGF-beta type II receptor. Thus, the inverse PCR with highly overlapped primers could be an easy way to isolate the promoter regions of many genes.     

Prognostic value of genomic damage in non-small-cell lung cancer

Genomic alterations have been analysed in 65 non-small-cell lung cancer (NSCLC) tissue samples by using the arbitrarily primed polymerase chain reaction (AP-PCR), which is a PCR-based genomic fingerprinting. We have shown that AP-PCR may be applied as a useful and feasible practical method for detection of the genomic alterations that accompany malignancy in NSCLC. Genomic changes detected by us consisted of: allelic losses or gains in anonymous DNA sequences, homozygously deleted DNA sequences and polymorphic DNA sequences. According to these genomic changes, lung tumours evaluated in the present study have been scored into three groups: low, moderate and high genomic damage tumours. The aim of this study was to investigate the effect of genomic damage on patient survival. Survival analysis was carried out in 51 NSCLC patients. Our results revealed that high genomic damage patients showed a poorer prognosis than those with low or moderate genomic damage (P = 0.038). Multivariate Cox regression analysis showed that patients with higher genomic alterations displayed an adjusted-by-stage risk ratio 4.26 times higher than the remaining patients (95% CI = 1.03-17.54). We can conclude that genomic damage has an independent prognostic value of poor clinical evolution in NSCLC.     

Identification of 12p as a region of frequent deletion in advanced prostate cancer

The identification of homozygous deletions in malignant tissue has been a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the PCR to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes such as BRCA2 and PTEN. Twenty RDA clones were generated by comparing genomic DNA from a prostate cancer xenograft to the same patient's normal kidney DNA. Southern blot analysis of the tester and driver and of normal and xenograft DNA, using the differential products as probes, showed the homozygous deletion in 16 of 20 RDA clones. The sequence of one of the differential products overlapped HSU59962, a genomic GenBank sequence on chromosome 12p12-13. Multiplex PCR of the xenograft DNA using polymorphic repeats mapped the deletion to a 1-5-cM region on 12p. Genomic DNA isolated from a panel of cryostat microdissected metastatic prostate adenocarcinomas/normal pairs was screened for loss of heterozygosity using the same polymorphic repeats. Loss of heterozygosity was demonstrated in 9 (47%) of 19 patients. This region may contain, or lie in close proximity to, tumor suppressor genes important in the progression and/or initiation of prostate cancer.     

Two-dimensional separations of the genome and proteome of neuroblastoma cells

Two-dimensional (2-D) electrophoretic methods have been available that allow separation of the protein constituents of a cell population. It has also become feasible to electrophoretically separate in two dimensions and to display DNA fragments derived from genomic digests. Through the appropriate choice of restriction enzymes, the functional component of the genome that encompasses CpG islands can be preferentially visualized in 2-D gels. The same computerized approach for the analysis of 2-D patterns can be applied to investigations at either the protein or DNA levels. Our group has utilized 2-D electrophoresis to investigate both protein and DNA changes in cancer. The emphasis to date has been on the identification of proteins, the abundance of which is related to specific biological features of the tumors analyzed and of DNA fragments encompassed in genomic amplifications, as the latter commonly contain growth-related genes. Findings derived from our analysis of neuroblastoma tumors and cell lines using 2-D approaches are reviewed. Data for four proteins observed in 2-D gels are presented because of our demonstrated association of these proteins with differentiation and proliferation properties of neuroblastoma. At the genomic level, the detection of amplifications using 2-D gels has necessitated an understanding of the variability displayed by multi-copy genomic fragments, which we have accomplished to a large part and which we present. An important benefit of 2-D approaches is the efficiency of scale and the ease with which abundant proteins or multicopy genomic fragments can be detected, identified and quantitatively analyzed.     

Introduction to the techniques of molecular biology

The purpose of this chapter is to outline some of the common recombinant DNA methods in use today. These techniques are usually employed to isolate a defined portion of the genome, mostly a gene, from an organism or tissue of interest and, thereafter, to characterize the structure and function of this genetic material. To isolate a gene, genomic DNA is extracted from a selected tissue. For a better handling the relatively large DNA molecules are cut into a mixture of fragments by restriction endonucleases. The fragments are then separated from each other according to their size by gel electrophoresis. A procedure called Southern blotting is used to verify the presence of the desired gene in one of the DNA fragments separated on an agarose gel. The DNA fragments are transferred from the gel to a filter whereby the original fragment pattern is maintained. Then, a single-stranded DNA or RNA probe specific for the gene to be isolated is hybridized to its target fragments fixed to the filter. A radioactive or fluorescent tag is attached to the probe for subsequent identification. In cases where only transcribed sequences are to be isolated cytoplasmic messenger RNA (mRNA) is prepared instead of DNA. Analysis of RNA by a technique similar to Southern blotting is termed Northern blotting. Preservation of DNA sequences is usually achieved by DNA cloning. DNA cloning involves the insertion of a DNA fragment into a DNA vector and the stable incorporation of the recombinant DNA into a suitable host. Propagation of the host facilitates the amplification of the recombinant DNA for subsequent analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of DNA fingerprinting to obstetrics and gynecology

Restriction fragment length polymorphisms (RFLP) are variations in the size of restriction fragments of genomic DNA that hybridize to specific probes. They are the consequence of changes in primary DNA sequences, most of which result from small-scale changes in DNA. Recently minisatellite DNA probes that detect many regions of great variability within the human genome have been described. Minisatellite probes consist of multiple repeated copies of a common 10-15 base pair core sequence. On hybridization to restriction enzyme digests of human DNA, they simultaneously detect many highly polymorphic minisatellites at different loci in the genome, and produce band patterns that are individual specific. The band patterns are called "DNA fingerprints" or "DNA barcode" which can be used for individual identification on forensic and legal medicine. In addition to forensic and legal medicine, DNA fingerprinting can be used in both basic research and clinical examination of obstetrics and gynecology. The RFLP bands in DNA fingerprinting are inherited as single Mendelian co-dominants and we can use such minisatellite DNA probe for the determination of zygosity in multiple pregnancy. This probe can be used for the determination of androgenesis as a cause of complete hydatidiform mole. Each polymorphic band in molar tissues could be identified as being of paternal but not maternal origin. Some polymorphic bands of paternal origin were not observed in molar tissues, indicating that endoreduplication of a normal haploid sperm or fertilization by dispermy to an anuclear oocyte with no effective genome could be the cause of complete hydatidiform mole (androgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and identification of DNA fragments frequently deleted from tumor cell lines of human esophageal cancer

OBJECTIVE: To clone tumor suppressor genes associated with human esophageal cancer (EC). METHODS: DNA fragments deleted from cell lines of esophageal cancer EC8712 and EC8733 were isolated by using modified genomic substractive hybridization. Twenty EC tissues paired with adjacent normal mucosa were analysed by Southern blot and PCR with these fragments as probes. RESULTS: Seven DNA fragments deleted from EC cell lines obtained were named as 12H1, 12H2, 33H3, 33H4, 12B1, 33B2 and 33B3. Sequencing revealed their size of 512bp, 428bp, 509bp, 355bp, 680bp (partially sequencing), 519bp and 425bp, respectively. 12H1 and 33H3 showed high homology (98%) to a new repeated sequence in human genome. 33H4 displayed 87% nucleotide identity with human alphoid-like repetitive sequence located on chromosome 17. 12B1 was homologous (90%) to LINE 1 transoposon containing two open reading frames. 12H2, 33B2 and 33B3 were novel sequences. These DNA fragments were deleted in 20%-60% of EC tissues and in 10%-20% of adjacent mucosa. CONCLUSIONS: Loss of 33H4 may be accompanied by deletion of an adjacent gene. 12B1 as transposon may suppress the tumorigenesis by activating suppressor genes or/and by inactivating oncogenes. 12H1/33H3, 12H2, 33B2 and 33B3 may be candidates of tumor suppressor genes associated with the development and the progression of esophageal cancer.     

Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid

We have systematically isolated and characterized DNA containing large CTG (n > 7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.     

Large-scale identification of virulence genes from Streptococcus pneumoniae

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.     

Identification of genes controlling the transition of Salmonella typhimurium bacteria to a non-culturable state

Mutants of Salmonella typhimurium with an impaired process of transition to the nonculturable state were tainted. Mutants were divided into four phenotypic groups. In four mutants (representatives of each phenotypic group), genes with TnPhoA transposon insertions were cloned; these insertions caused a disturbance in the process of mutant cell transition to the nonculturable state. Nucleotide sequences of mutant gene fragments were determined. Comparison of nucleotide sequences obtained with a data bank on DNA nucleotide sequences of enterobacterial genomes allowed the identification of four genes involved in the control of nonculturable form generation in salmonellae.     

Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein

The genes encoding the two different subunits of the electron transfer flavoprotein (ETF) from the methylotrophic bacterium W3A1 have been identified, cloned, and sequenced. A 0.8-kilobase pair DNA fragment was generated for use as a molecular probe by the amplification of genomic sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the NH2-terminal amino acid sequences determined for the ETF subunits purified from W3A1. The screening of a partial genomic minilibrary containing size-selected BamHI-SalI fragments using this probe identified a 2.2-kilobase pair insert containing the complete coding sequences for both W3A1 ETF subunits. The genes are arranged in tandem in the genomic DNA with only 2 bases between the TAG translation termination codon of the small subunit and the ATG translation initiation codon of the large subunit. The deduced amino acid sequences of each of the W3A1 ETF subunits exhibit only approximately 30% identity with the corresponding subunits of the ETF from human, rat, and Paracoccus denitrificans, which as a group are greater than 50% identical. Thus, the ETF from W3A1 may exhibit some unique structural features that, like other differences in some of its physical and functional properties, may distinguish this ETF from others in this family. A highly homologous region near the COOH terminus of the large subunit in all the ETF proteins was found to contain a sequence that matches in several ways the ADP-binding motif of flavoproteins and other dinucleotide-binding proteins, suggesting that the large subunit forms a portion of the FAD (or AMP) binding site in these proteins. Under control of the tac promoter, the cloned ETF subunit genes were co-expressed in Escherichia coli producing the heterodimeric holoprotein with physical, spectral, and electron-accepting properties essentially identical to the ETF isolated from W3A1. The recombinant ETF serves as the electron acceptor for W3A1 trimethylamine dehydrogenase in vitro, accumulating as the air-stable anionic semiquinone in the presence of excess trimethylamine. Fully reduced ETF could not be obtained even after prolonged enzymatic reduction.     

Characteristics of two salmonid repetitive DNA families in rainbow trout, Oncorhynchus mykiss

DNA sequence and genomic location of two repetitive DNA families in rainbow trout (Oncorhynchus mykiss) were investigated to develop molecular markers for chromosome identification. DNA fragments with sequences similar to the tandem and interspersed elements described in other salmonids were isolated. One clone showed differential hybridization to 12 pairs of chromosomes and should be a useful marker for physical mapping.     

Representational difference analysis of a committed myeloid progenitor cell line reveals evidence for bilineage potential

In this study we have sought to characterize a committed myeloid progenitor cell line in an attempt to isolate general factors that may promote differentiation. We used cDNA representational difference analysis (RDA), which allows analysis of differential gene expression, to compare EML and EPRO cells. We have isolated nine differentially expressed cDNA fragments as confirmed by slot blot, Northern, and PCR analysis. Three of nine sequences appear to be novel whereas the identity of the remaining fragments suggested that the EPRO cell line is multipotent. Among the isolated sequences were eosinophilic, monocytic, and neutrophilic specific genes. Therefore, we tested the ability of EPRO cells to differentiate along multiple myeloid lineages and found that EPRO cells exhibited morphologic maturation into either monocyte/macrophages or neutrophils, but not eosinophils. Furthermore, when EPRO cells were exposed to ATRA, neutrophil specific genes were induced, whereas monocytic markers were induced by phorbol ester treatment. This study highlights the use of cDNA RDA in conjunction with the EML/EPRO cell line to isolate markers associated with macrophage and neutrophil differentiation and establishes the usefulness of this system in the search for factors involved in myeloid commitment.     

Characterization of oligonucleotide probes for the identification of Acinetobacter spp., A. baumannii and Acinetobacter genomic species 3

The 16S-23S intergenic spacer regions of four Acinetobacter genomic species belonging to the A. calcoaceticus-A. baumannii (Acb) complex, i.e. genomic species 1 (A. calcoaceticus), genomic species 2 (A. baumannii), genomic species 3 and Tjernberg and Ursing (TU) genomic species 13, have been cloned and sequenced. Sequence analysis led to the discovery of a single copy of IIe and Ala tRNA genes within each spacer. Sequence comparison allowed the identification of a 192-base-pair long highly conserved sequence between the 3' end of the 16S rRNA and the 5' end of the tRNA(Ala) genes. Moreover, two short regions, which were specific to, respectively, genomic species 2 and 3, could be identified. Oligonucleotides corresponding to these sequences were constructed and tested for the ability to hybridize with chromosomal DNA extracted from Acinetobacter belonging to different genomic species and with chromosomal DNA of other bacterial genera. One of these oligonucleotides was demonstrated to be useful as a sensitive and specific probe for A. baumannii. A less sensitive probe for Acinetobacter genomic species 3 was also developed.     

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.