Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.     

Expressed sequence tags from a NaCl-treated Suaeda salsa cDNA library

Past efforts to improve plant tolerance to osmotic stress have had limited success owing to the genetic complexity of stress responses. The first step towards cataloging and categorizing genetically complex abotic stress responses is the rapid discovery of genes by the large-scale partial sequencing of randomly selected cDNA clones or expressed sequence tags (ESTs). Suaeda salsa, which can survive seawater-level salinity, is a favorite halophytic model for salt tolerant research. We constructed a NaCl-treated cDNA library of Suaeda salsa and sequenced 1048 randomly selected clones, out of which 1016 clones produced readable sequences (773 showed homology to previously identified genes, 227 matched unknown protein coding regions, 16 anomalous sequences or sequences of bacterial origin were excluded from further analysis). By sequence analysis we identified 492 unique clones: 315 showed homology to previously identified genes, 177 matched unknown protein coding regions (101 of which have been found before in other organisms and 76 are completely novel). All our EST data are available on the Internet. We believe that our dbEST and the associated DNA materials will be a useful source to scientists engaging in stress-tolerance study.     

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA ULVA LINZA (CHLOROPHYTA)1

There is a general lack of genomic information available for chlorophyte seaweed genera such as Ulva, and in particular there is no information concerning the genes that contribute to adhesion and cell wall biosynthesis for this organism. Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery and characterization of expression patterns. In this study, a cDNA library was created from sporulating tissue of Ulva linza L. Initially, 650 ESTs were randomly selected from a cDNA library and sequenced from their 5′ ends to obtain an indication of the level of redundancy of the library (21%). The library was normalized to enrich for rarer sequences, and a further 1920 ESTs were sequenced. These sequences were subjected to contig assembly that resulted in a unigene set of approximately 1104 ESTs. Forty‐eight percent of these sequences exhibited significant similarity to sequences in the databases. Phylogenetic comparisons are made between selected sequences with similarity in the databases to proteins involved in aspects of extracellular matrix/cell wall assembly and adhesion.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

Construction of Rice cDNA Library and Partial Sequence Analysis of 100 Randomly Selected cDNA

A cDNA library of rice (Oryza sativa ssp. indica cv. "Guangluai 4") etiolated shoot was constructed using Lambda ZAP Ⅱ vector. After analysing the partial sequences of 100 randomly selected clones and database comparison to rice and other plants, 13 % cDNA clones could be identified and 12 % cDNAs had high degree of sequence similarity to partial sequence from rice or other species, whose function is still unknown. The remaining 75% cDNAs showed little or no similarity to genes in the database and might represent novel genes. It demonstrates the suitability of this library for large-scale sequencing from which more information of functional genes will result.     

Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages

Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.     

Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis

The epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. Small non-coding RNAs (sncRNAs), especially microRNAs (miRNAs), can participate in the regulation of various physiological pathways. However, their abundance and whether they are involved in the regulation of gene expression in the human epididymis are unknown. By adopting the Solexa deep sequencing approach, we systematically investigated the sncRNAs in the adult human epididymis. A total of 4903 unique sequences representing 527 known miRNA were discovered. Eighteen novel miRNA genes encoding 23 mature miRNAs were also identified and the expression of some of them was confirmed by qRT-PCR. The presence of Piwi-interacting RNAs (piRNAs) in the library also adds to the diversity of the sncRNA population in the human epididymis. This research will contribute to a preliminary database for their functional study in male reproductive system.     

A proteomic approach to identify phosphoproteins encoded by cDNA libraries

We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.     

Method enabling fast partial sequencing of cDNA clones

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.     

Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein.

A cDNA expression library in lambda gt11 prepared from cDNA derived of seminal vesicle tissue was screened by means of monospecific rabbit anti-aSFP IgG. The sequence of clone pTF21, containing an insert of 668 bp comprised an open reading frame from position 7 to 411 terminated by two stop codons. From this sequence a protein of 134 amino acid residues can be deduced. The mature aSFP was preceded by a signal peptide of 20 amino acids length. The protein sequence contains no signal for N-glycosylation. The molecular weight calculated from the amino acid sequence is 12922 Da. The start codon ATG is part of the sequence AAGATGA which fulfills the criteria of an initiation consensus sequence. The coding region was followed by 257bp of the complete 3'-untranslated region (3'UTR). A putative polyadenylation signal AATAAT, although not of the standard type, is observed at position 650. According to Northern analysis, aSFP mRNA is expressed in seminal vesicle tissue, ampulla and weakly in tissue of epididymis, but not in testis or other bovine tissue. aSFP is specified by a single copy gene. Attempts to detect homologies to known protein sequences were not successful.     

Exploring the diversity of myxobacteria in a soil niche by myxobacteria-specific primers and probes

The diversity of myxobacteria in a soil niche was explored using culture-dependent and -independent methods. Conventional cultivation for bacteriolytic myxobacteria produced six types of myxobacteria, which were identified as two Myxococcus spp., two Corallococcus spp., a Cystobacter sp. and a Nannocysts sp. Hybridization analysis of the soil bacterial 16S rRNA gene library with myxobacteria-specific probes revealed that myxobacteria accounted for less than 1% in the bacterial community. A Cystobacterineae 16S rRNA genes-rich library was further established from the soil DNA by polymerase chain reaction amplification with a Cystobacterineae-specific primer combined with a universal bacterial primer. Screening of the special library using Cystobacterineae- and Sorangineae-specific probes produced approximately 45% and 3% positive signals respectively. Sixty-four positive clones were randomly selected for sequencing. Except three repeats, the sequences were diverse ranging from 0.3% to 21.3%, and homologous with the known myxobacteria at 77.6-99.8%, including 57 in Cystobacterineae, one close to Nannocystis and three much more distant from the known myxobacteria. The sequences in the Cystobacterineae can further be divided into at least 12 groups, of which most were unreported. The results suggest that myxobacteria in nature are much more diverse than were ever known, even in one soil niche.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.     

CHARACTERIZATION OF EXPRESSED SEQUENCE TAGS GENERATED FROM SKIN CDNA CLONES OF Equus caballus BY SINGLE PASS SEQUENCING

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10⁵ plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.