Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology

To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes-lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)-with unknown functions in the epididymis. The novel gene, designated Serpina1f (serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except forLcp1. Testosterone (T) restored these reduced expressions, except forTeddm1; however, this restoration was not observed with 17 beta-estradiol (E2). Administration of T and E2 combination recovered the Serpina1f mRNA concentration; this recovery was also observed with T alone. However, the recovery of Cuzd1and Wfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed the Cuzd1, Wfdc16, and Serpina1f mRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.     

Identification and characterization of an epididymis-specific gene,Ces7

Carboxylesterases （CEs） represent a multigene family of serine-dependent enzymes. Male-dependent CEs are over-expressed in the male reproductive tract of different animal species （bivalve mollusks, fruit-flies, and mammals）. Here, a novel rat epididymis-specific gene named Ces7 was cloned and characterized. It was a novel member of CE family, which was mainly expressed and secreted to the lumens of the corpus and cauda epididymis. CES7 protein was highly glycosylated as other mammalian CEs. Furthermore, Ces7 increased with age growth until sex maturation and then maintained at high level. CES7 might be one of the major CEs in male reproductive tract and contribute to the sperm fertilization.     

Discovery and characterization of new epididymis-specific beta-defensins in mice

The male urogenital tract epithelium is exposed to several pathogens, but only a few are potent enough to cause infection in a healthy individual. The exact mechanisms that protect the male reproductive tract from ascending pathogenic micro-organisms are still poorly characterized. We recently reported a method to identify novel epididymis-specific genes by analyzing the expressed sequence tags (ESTs) present in the mouse epididymal cDNA library of the UniGene collection at National Center for Biotechnology Information (NCBI). In the present study, we discovered in silico two novel epididymal genes: the beta-defensins Defb41 and Defb42. The full-length cDNAs for the genes were acquired by the RT-PCR and 5'-RACE approaches and were subsequently sequenced. Q-RT-PCR and in situ hybridization revealed Defb41 and Defb42 to be expressed mainly in the proximal caput. The expression of both defensins was found to be regulated by androgens. Based on their structure and expression pattern, Defb41 and Defb42 are suggested to have a role in the antimicrobial protection of sperm and urogenital tract epithelia.     

Identification and characterization of antibodies elicited by human cystatin C fragment

Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. One of rather atypical, occurring at a young age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real, lethal threat to some populations and any work on factors that can affect possible influencing hCC aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue 60‐70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in rabbit immunization. As a result, specific anti‐hCC antibodies were found in both rabbit sera. Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The obtained antibodies were characterized, and their influence on the aggregation propensity of the hCC molecules was evaluated. The antibodies turned out not to have any significant influence on the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other hCC fragments could lead to elaboration of effective treatment against HCCAA.     

Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri

Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear.     

Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice

Defensins comprise a family of cationic antimicrobial peptides that are characterized by the presence of six conserved cysteine residues. We identified two novel human beta-defensin (hBD) isoforms by mining the public human genomic sequences. The predicted peptides conserve the six-cysteine motif identical with hBD-4, termed hBD-5 and hBD-6. We also evaluated the characteristics of the mouse homologs of hBD-5, hBD-6, and HE2beta1, termed mouse beta-defensin (mBD)-12, mBD-11, and mouse EP2e (mEP2e). The mBD-12 synthetic peptide showed salt-dependent antimicrobial activity. We demonstrate the epididymis-specific expression pattern of hBD-5, hBD-6, mBD-11, mBD-12, and mEP2e. In situ hybridization revealed mBD-11, mBD-12, and mEP2e expression in the columnar epithelium of the caput epididymis, contrasting with the predominant expression of mBD-3 in the capsule or septum of the whole epididymis. In addition, the regional specificity of mBD-11, mBD-12, and mEP2e was somewhat overlapping, but not identical, in the caput epididymis, suggesting that specific regulation may work for each member of the beta-defensin family. Our findings indicated that multiple beta-defensin isoforms specifically and cooperatively contribute to the innate immunity of the urogenital system.     

Soluble expression and characterization of a mouse epididymis-specific protein lipocalin6

Mouse lipocalin6 (mLcn6) was recently identified to be specifically expressed in the epididymis and speculated to may play a role in sperm maturation. However, further studies were hindered due to the bottleneck to obtain enough recombinant mLcn6 proteins. In this article, GB1 tag was successfully applied to improve the soluble expression of mLcn6. Thermal unfolding experiments demonstrate that GB1 can enhance the structural stability of mLcn6. Fluorescence spectroscopy experiments show that mLcn6 prepared according to our procedure has high affinities to both retinoic acid (K_d = 810 nM) and retinol (K_d = 210 nM). In conclusion, soluble, stable and active mLcn6 was recombinantly prepared with the help of the GB1 tag, which will facilitate the structural and functional studies of mLcn6.     

Identification and characterization of lineage-specific genes in Populus trichocarpa

Species- or lineage-specific genes can facilitate studying the unique characteristics of biological processes. Updated genome sequences in Populus trichocarpa were screened against thirty newly sequenced or resequenced plant genomes to identify a set of species-specific genes (PtSS). Forty PtSS genes have been isolated with no similarity to any sequence outside the P. trichocarpa genome, therefore have no annotated functions. Protein motif, intron/exon features, subcellular localization and gene expression were analyzed in these PtSS genes. Results reflect their basic genic characters, expression analysis and primary function exploration might provide insight to their possible involvements in lineage specific biological process in woody plants.     

Identification and physical characterization of yeast maltase structural genes

Summary Each of at least five unlinked MAL loci (MAL1 through MAL4 and MAL6) on the yeast genome controls the ability to synthesize an inducible -D-glucosidase (maltase). A subcloned fragment of the coding sequence of the MAL6 maltase structural gene was used as a hybridization probe to investigate the physical structure of the family of MAL structural genes in the genomes of different Saccharomyces strains. Mal⁺ strains, each carrying a genetically defined MAL locus, were crossed with a Mal^- strain and the segregation behavior of the functional locus and of sequences complementary to the maltase structural gene at that locus analyzed. The maltase structural gene sequences of each MAL locus were detected by Southern blot hybridization using BamH1 digests of genomic DNA of the meiotic products. This restriction enzyme was previously shown to cleave outside the confines of the MAL6 locus.The results of such experiments indicate that each MAL locus encompasses at least one maltase structural gene sequence homologous to that of MAL6, that yeast strains that lack functional MAL loci may or may not contain the corresponding maltase structural gene sequence, that the MAL1 maltase structural gene sequence or one of its alleles can be detected in all laboratory yeast strains examined and that each MAL locus can be identified as a characteristic BamH1 fragment of genomic DNA which includes a maltase structural gene.Yeast strains vary in the number of maltase structural gene sequences that they carry. By using the approach described in this report, the ones corresponding to the different functional MAL loci and residing within a BamH1 generated restriction fragment can be identified.     

Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.     

Identification and characterization of putative CIPK genes in maize

Calcium(Ca) plays a crucial role as a second messenger in intracellular signaling elicited by developmental and environmental cues. Calcineurin B-like proteins(CBLs) and their target proteins,CBL-interacting protein kinases(CIPKs) have emerged as a key Ca~(2+)-mediated signaling network in response to stresses in plants.Bioinformatic analysis was used to identify 43 putative ZmCIPK(Zea mays CIPK) genes in the genome of maize inbred line B73.Based on gene structures,these ZmCIPKs were divided into intron-...     

Identification and characterization of zebrafish ocular formation genes.

To study genes that are specifically expressed in the eyes, we employed microarray and in situ hybridization analyses to identify and characterize differentially expressed ocular genes in eyeless masterblind (mbl-/-) zebrafish (Danio rerio). Among 70 differentially expressed genes in the mbl-/- mutant identified by microarray analysis, 8 down-regulated genes were characterized, including 4 eye-specific genes, opsin 1 short-wave-sensitive 1 (opn1sw1), crystallinbetaa1b (cryba1b), crystallinbetaa2b (cryba2b), and crystallingamma M2d3 (crygm2d3); 2 eye and brain genes, ATPase, H+ transporting, lysosomal, V0 subunit c (atp6v0c) and basic leucine zipper and W2 domains 1a (bzw1a); and 2 constitutive genes, heat shock protein 8 (hspa8) and ribosomal protein L7a (rpl7a). In situ hybridization experiments confirmed down-regulation of these 8 ocular formation genes in mbl-/- zebrafish and showed their ocular and dynamic temporal expression patterns during zebrafish early development. Further, an automated literature analysis of the 70 differentially expressed genes identified a sub-network of genes with known associations, either with each other or with ocular structures or development, and shows how this study contributes to the current body of knowledge.