Isolation of Unknown Genes from Human Bone Marrow by Differental Screening and Single-Pass cDNA Sequences Determination

A cDNA sequencing project was initiated to characterize gene expression in human bone marrow and develop strategies to isolate novel genes. Forty-eight random cDNAs from total human bone marrow were subjected to single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed no similarity to known sequences. Information from DNA sequence analysis was used to develop a differential prescreen to subtract unwanted cDNAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negative with a complex probe were subject to single-pass DNA sequence determination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA expression analysis using 25 different human leukemic cell lines. Of 13 unknown cDNAs tested, 10 were expressed in all cell types tested and 3 revealed a hematopoietic lineage-restricted expression pattern. Interestingly, while a total of only 96 bone marrow cDNAs were sequenced, 31 of these cDNAs represent sequences from unknown genes and 12 showed significant similarities to sequences in the data bases. One cDNA revealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may represent a previously unknown kinase. Differential screening techniques coupled with single-pass cDNA sequence analysis may prove to be a powerful and simple technique to examine developmental gene expression

NCBI BLASTN Stage 1 in Reconfigurable Hardware

Recent advances in DNA sequencing have resulted in several terabytes of DNA sequences. These sequences themselves are not informative. Biologists usually perform comparative analysis of DNA queries against these large terabyte databases for the purpose of developing hypotheses pertaining to function and relation. This is typically done using software on a general multiprocessor. However, these data sets far exceed the capabilities of the modern processor and performing sequence similarity analysis is increasingly becoming less efficient. There is an urgent need for more efficient ways of querying large DNA sequences for sequence similarities. Here, we describe an FPGA-based hardware solution that implements Stage 1 of NCBI BLASTN, a commonly used sequence analysis application

Molecular Markers and Their Optimization: Addressing the Problems of Nonhomology Using Decapod COI Gene

Advancements in DNA sequencing and computational technologies influenced almost all areas of biological sciences. DNA barcoding technology employed for generating nucleotide sequences (DNA barcodes) from standard gene region(s) is capable of resolving the complexities caused due to morphological characters. Thus, they complement taxonomy, population analysis, and phylogenetic and evolutionary studies. DNA barcodes are also utilized for species identification from eggs, larvae, and commercial products. Sequence similarity search using Basic Local Alignment Search Tool (BLAST) is the most reliable and widely used strategy for characterizing newly generated sequences. Similarity searches identify “homologous” gene sequence(s) for query sequence(s) by statistical calculations and provide identity scores. However, DNA barcoding relies on diverse DNA regions which differ considerably among taxa. Even, region-specific variations within barcode sequences from a single gene leading to “nonhomology” have been reported. This causes complications in specimen identification, population analysis, phylogeny, evolution, and allied studies. Hence, the selection of appropriate barcode region(s) homologous to organism of interest is inevitable. Such complications could be avoided using standardized barcode regions sequenced using optimized primers. This chapter discusses about the potential problems encountered due to the unknown/unintentional/intentional use of nonhomologous barcode regions and the need for primer optimization

PhD

dissertationThe vertebrate pro-opiomelanocortin (POMC) gene codes for the large protein precursor to a number of small peptides with highly conserved sequence, including adrenocorticotropin (ACTH), melanocyte stimulating hormone (MSH), and $/beta$-endorphin. previous evidence suggests that structurally and possibly functionally related sequences (homologs) are present in unicellular organisms, and that these may be encoded within a similarly organized genes. This work attempted to identify a POMC homolog in the protozoan, Tetrahymena pyriformis, and in fission yeast, Saccharomyces cerevisiae. Using two different equilibrium-type radioimmunoassays with polyclonal antibodies, I initially identified ACTH-like immunoactivity in extracts of both species. However, the same extracts showed no reaction in an immunoradiometric assay requiring greater structural similarity to vertebrate ACTH. The low concentration meant purification of adequate amounts of material to sequence was impractical. Southern analysis of Tetrahymena genomic DNA was unfruitful in identifying POMC-related sequences. However, yeast genomic DNA showed hybridizing bands when probed at low stringency with the rat POMC gene. Screening a yeast genomic DNA library under low stringency resulted in identification of one of the DNA sequences responsible. Analysis of hybridizing DNA showed a long open reading frame coding for a putative protein of 610 amino acids, showing 18% identity and 45% chemical similarity to the rat POMC precursor, with 30% identity to the vertebrate ACTH sequence. There was no significant homology to other vertebrate POMC-derived peptides, and the protein showed no features suggesting cleavage into smaller peptides. The presence of a leucine zipper motif and multiple phosphorylation sites indicated an ability to dimerize and possibly bind DNA. A second open reading frame was also found on the opposite DNA strand 500 bases upstream, showing strong homology to DNA-unwinding proteins. A poly(dA-dT) stretch between both genes may function as a common regulatory sequence. Northern analysis indicated the ORF1gene was expressed. However, deletion and overexpression analysis showed it was not likely to be responsible for the measured immunoactivity, that it was not essential, and that it had no effect on growth rate under normal aerobic conditions. Significance of the sequence similarity to POMC and possible functions are discussed

DNA Sequences of RAPD Fragments in the Egyptian cotton Gossypium barbadense

Random Amplified Polymorphic DNAs (RAPDs) is a DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. Despite the fact that the RAPD technique has become a very powerful tool and has found use in numerous applications, yet, the nature of molecular variation(s) uncovered by the RAPD technique is still unclear. The aim of the following study, therefore, was to investigate the molecular nature of RAPD DNA fragments in four Gossypium barbadense cultivars. Five RAPD DNA fragments, generated by improved RAPD-PCR technique, and representing polymorphic and nonpolymorphic bands were analyzed at the molecular level using DNA sequence analysis. Nonpolymorphic RAPD DNA fragments showed homologies to previously characterized plant structural genes. Comparative nucleotide sequence analysis of two comigrating nonpolymorphic fragments revealed that these two DNA sequences are highly similar to each other, indicating that similarity of fragment size is a good predicator of homology. Polymorphic RAPD DNA fragments, on the other hand, showed homologies to middle and high-repetitive DNA sequences. These results promote the initiative to integrate these RAPD markers in cotton breeding applications, and DNA fingerprinting. (African Journal of Biotechnology: 2003 2(5): 129-132

Comparative insect mitochondrial genomes: Differences despite conserved genome synteny

We present a comparative analysis of select insect mitochondrial DNA (mtDNA) representing four insect orders (Diptera, Hymenoptera, Orthoptera and Coleoptera) consisting of 12 different species inan effort to study a common set of genes and to understand the evolution of mitochondrial genome. A functional analysis of mitochondrial genomes was carried out using ERGO bioinformatics suite. To compare the similarity between closely related insect mitochondrial genome sequences, dot-plotcomparisons of sequences were performed. LSU and SSU rRNA sequences were used to construct a phylogenetic tree to determine the relationship among four insect orders. LSU rRNA sequences yieldeda tree with branching patterns reflecting the expected pattern as insect species belonging to different orders were put into separate clades. Based on the sequence similarity, insect species belonging tofour different orders in general appear to be closely related. However, a comparative and functional analysis of insect mitochondria sequences revealed differences in gene organization of mtDNA. Although tRNA species were identical in most species of insects, their position and the transcription orientations were different, reflecting differential transcriptions. Based on this study we conclude that,although the gene types are very similar across these insect orders, significant differences in GC content perhaps suggest multiple mitochondrial ancestor

Molecular Identification of Soil Bacteria by 16srDNA Sequence

In this current study, 16S rDNA (genotypic) identification technique is focused on identification of conventionally unidentifiable isolates those are unevaluated in isolated by employing molecular techniques and Bioinformatics in uploading and retrieving isolate gene sequences which are rapid, reliable and accurate in differentiation of various soils isolates. This study is an automaton of 16Sr DNA gene sequence that allows a queue comparison analysis of published sequences deposited in the microbial genome database was used. Polymerase chain reaction (PCR) amplification of 16SrDNA gene using the consensus bacterial primer and separation of the resulting polymer chain reaction amplicon  by cloning, temperature gradient electrophoresis are major ecological techniques that are used in the description of soil bacteria. The isolated gene was cloned using PTZ57r or T cloning Vector amplified using 16SF and 16SR primer transformed in DH5? Cells resulting PCs 16s Plasmid hybrid. The primer 16S F2 obtained from M13 forward primer was used and aligned using BLAST and submitted to EMBL+GENEBANK+DDBJ+ PDB. 99% similarity was observed and later it was analyzed with the existing sequence in ribosomal database project II.  RDP classifier was used for confirmation with 100 % similarity. The bacteria were identified as Burkholderia cenocepacia  when the  sequence was submitted and retrieved via the World Wide Web and new sequence compared with those held in the database  using the basic local alignment tool (BLAST). A segment of 734 out of 736 nucleotide of 16S rDNA gene of Burholderia Cenocepacia is the region of choice for primer construction because of proximity that provides a successful discrimination in strains of Burholderia Cenocepacia in soil. 16S rDNA gene account to 99%  similarity score in molecular typing and identification of bacteria which concerns deposition of sequences into established microbial genomic database Key Words: Burkholderi; Bacterial transformation; Characterization; DNA based technique

Comparison of Fusarium oxysporum fsp lycopersici races 1, 2 and 3, and f.sp radicis lycopersici based on the sequences of fragments of the ribosomal DNA intergenic spacer region

Sequence analysis of genomic fragments from the intergenic spacer region from three isolates of Fusarium oxysporum fsp lycoperisci and fsp radicis lycopersici was carried out using the big dyeterminator sequencing procedure. Two conditions of the DNA templates were also evaluated for their influence on the outcome of the terminator reaction. Results showed that sequencing using the PCR products of M13 primer reaction with either direct E. coli colony, (condition 1) or purified plasmid DNA as templates (condition 2), were successful and the sequences of the cloned IGS fragments were the same indicating that time and cost could be minimized by excluding the plasmidpurification steps. Based on the sequence analysis of the IGS fragment of race 1 (kis-1a) (ca. 638 bp including the forward and reverse primers sequences) it is observed that there is at least 95% similarity between the F. oxysporum races 1, 2, 3, and rly. Using the BioEdit sequence analysis program, there are 14 conserved regions with the longest continuous consensus segment being between nucleotide position number 1 and 129. Region 2 has 18 segment length (164-181), whileregion 3 is the shortest region with 15 segment length (183-197)

Improved alignment of nucleosome DNA sequences using a mixture model

DNA sequences that are present in nucleosomes have a preferential ∼10 bp periodicity of certain dinucleotide signals (1,2), but the overall sequence similarity of the nucleosomal DNA is weak, and traditional multiple sequence alignment tools fail to yield meaningful alignments. We develop a mixture model that characterizes the known dinucleotide periodicity probabilistically to improve the alignment of nucleosomal DNAs. We assume that a periodic dinucleotide signal of any type emits according to a probability distribution around a series of ‘hot spots’ that are equally spaced along nucleosomal DNA with 10 bp period, but with a 1 bp phase shift across the middle of the nucleosome. We model the three statistically most significant dinucleotide signals, AA/TT, GC and TA, simultaneously, while allowing phase shifts between the signals. The alignment is obtained by maximizing the likelihood of both Watson and Crick strands simultaneously. The resulting alignment of 177 chicken nucleosomal DNA sequences revealed that all 10 distinct dinucleotides are periodic, however, with only two distinct phases and varying intensity. By Fourier analysis, we show that our new alignment has enhanced periodicity and sequence identity compared with center alignment. The significance of the nucleosomal DNA sequence alignment is evaluated by comparing it with that obtained using the same model on non-nucleosomal sequences

Isolation and linkage mapping of NBS-LRR resistance gene analogs in red raspberry ( Rubus idaeus L.) and classification among 270 Rosaceae NBS-LRR genes

Plant R genes confer resistance to pathogens in a gene-for-gene mode. Seventy-five putative resistance gene analogs (RGAs) containing conserved domains were cloned from Rubus idaeus L. cv. ‘Latham' using degenerate primers based on RGAs identified in Rosaceae species. The sequences were compared to 195 RGA sequences identified from five Rosaceae family genera. Multiple sequence alignments showed high similarity at multiple nucleotide-binding site (NBS) motifs with homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR RNBSA-A motifs. The TIR sequences clustered separately from the non-TIR sequences with a bootstrap value of 76%. There were 11 clusters each of TIR and non-TIR type sequences of multiple genera with bootstrap values of more than 50%, including nine with values of more than 75% and seven of more than 90%. Polymorphic sequence characterized amplified region and cleaved amplified polymorphic sequence markers were developed for nine Rubus RGA sequences with eight placed on a red raspberry genetic linkage map. Phylogenetic analysis indicated four of the mapped sequences share sequence similarity to groupTIR I, while three others were spread in non-TIR groups. Of the 75 Rubus RGA sequences analyzed, members were placed in five TIR groups and six non-TIR groups. These group classifications closely matched those in 12 of 13 studies from which these sequences were derived. The analysis of related DNA sequences within plant families elucidates the evolutionary relationship and process involved in pest resistance development in plants. This information will aid in the understanding of R genes and their proliferation within plant genome