Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting genotoxins

During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK(+/-) mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown.     

Cloning, sequencing and expression of a sialidase gene from Clostridium sordellii G12

A 4.3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0.7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44,735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C. sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.     

Identification and expression profile of novel STAND gene Nwd2 in the mouse central nervous system

In the central nervous system (CNS), neurons need synaptic neurotransmitter release and cellular response for various cellular stress or environmental stimuli. To achieve these highly orchestrated cellular processes, neurons should drive the molecular mechanisms that govern and integrate complex signaling pathways. The signal transduction ATPases with numerous domains (STAND) family of proteins has been shown to play essential roles in diverse signal transduction mechanisms, including apoptosis and innate immunity. However, a comprehensive understanding of STAND genes remains lacking. Previously, we identified the NACHT and WD repeat domain-containing protein 1 (NWD1), a member of STAND family, in the regulation of the assembly of a giant multi-enzyme complex that enables efficient de novo purine biosynthesis during brain development. Here we identified the mouse Nwd2 gene, which is a paralog of Nwd1. A molecular phylogenetic analysis suggested that Nwd1 emerged during the early evolution of the animal kingdom, and that Nwd2 diverged in the process of Nwd1 duplication. RT-PCR and in situ hybridization analyses revealed the unique expression profile of Nwd2 in the developing and adult CNS. Unlike Nwd1, Nwd2 expression was primarily confined to neurons in the medial habenular nucleus, an essential modulating center for diverse psychological states, such as fear, anxiety, and drug addiction. In the adult brain, Nwd2 expression, albeit at a lower level, was also observed in some neuronal populations in the piriform cortex, hippocampus, and substantia nigra pars compacta. NWD2 might play a unique role in the signal transduction required for specific neuronal circuits, especially for cholinergic neurons in the habenula.     

Identification of a potential ovarian cancer stem cell gene expression profile from advanced stage papillary serous ovarian cancer

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.     

Identification and expression of the elongator protein 2 (Ajelp2) gene,a novel regeneration-related gene from the sea cucumber Apostichopus japonicus

Elongator proteins comprise six subunits (ELP1–ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5′ untranslated (UTR), a 526 bp 3′ UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it’s the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.     

Identification of gene expression profile of neural crest-derived cells isolated from submandibular glands of adult mice

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity.     

管纹艳虎天牛糖苷水解酶基因家族的鉴定和表达谱分析

昆虫糖苷水解酶（Glycoside hydrolase, GH）在寄主植物糖类化合物的水解过程中扮演着重要作用,但是在管纹艳虎天牛Rhaphuma horsfieldi中尚未有GH基因的报道。基于测序的转录组数据,本研究从管纹艳虎天牛中鉴定到7个RhorGHs家族：GH1、GH9、GH13、GH16、GH28、GH31和GH45,分别具有23、1、4、2、10、12和4个基因,其中23个基因具有全长序列。在进化分析中,管纹艳虎天牛不同GH家族的成员主要以物种特异的形式聚类,尤其是GH1和GH28家族。三级结构分析显示,RhorGH28家族的蛋白主要由β折叠组成,不同蛋白间具有高度保守的结构和与糖类化合物互作的关键氨基酸位点,包括质子供体、催化亲核残基和底物结合位点。在表达谱分析中,大部分RhorGHs基因主要在雌雄虫腹部特异或高表达,暗示其可能在与消化有关的肠道中表达。研究结果明确了管纹艳虎天牛GH家族及其数量,以及各家族的序列特征和进化关系,可为该天牛的寄主植物适应性机制研究提供借鉴。     

Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data

BACKGROUND: Flow cytometry of immunofluorescence and DNA content provides measures of cell-cycle-related gene expression (protein and/or epitope levels) for asynchronously growing cells. From these data, time-related expression through S phase can be directly measured. However, for G1, G2, and M phases, this information is unavailable. We present an objective method to model G1 and G2 kinetic expression from an estimate of a minimum biological unit of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. METHODS: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B1) distribution of p105-positive cells was used to model the B1 distribution of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. RESULTS: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mitotic levels were equivalent to the highest G2 levels. G1 modeling of B1 was less certain than that of G2 due to low levels of expression but demonstrated general feasibility. CONCLUSIONS: By this method, the upper and lower bounds of cyclin B1 expression could be estimated and kinetic expression through G1, G2, and M modeled. Together with direct measurements in S phase, expression of B1 throughout the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions.     

Identification and expression profile analysis of the protein kinase gene superfamily in maize development

Eukaryotic protein kinases (ePKs) evolved as a family of highly dynamic molecular switches that serve to orchestrate the activity of almost all cellular processes. Some of the functionally characterized ePKs from plants have been found to be components of signaling networks, such as those for the perception of biotic agents, light quality and quantity, plant hormones, and various adverse environmental conditions. To date, only a tiny fraction of plant ePKs have been functionally identified, and even fewer have been identified in maize [Zea mays (Zm)]. In this study, we have identified 1,241 PK-encoding genes in the maize genome. Phylogenetic analyses identified eight gene groups with considerable conservation among groups, and each group could be further divided into multiple families and/or subfamilies. Similar intron/exon structural patterns were observed in the same families/subfamilies, strongly supporting their close evolutionary relationship. Chromosome distribution and genetic analysis revealed that tandem duplications and segmental/whole-genome duplications might represent two of the major mechanisms contributing to the expansion of the PK superfamily in maize. The dynamic expression patterns of ZmPK genes across the 60 different developmental stages of 11 organs showed that some members of this superfamily exhibit tissue-specific expression, whereas others are more ubiquitously expressed, indicative of their important roles in performing diverse developmental and physiological functions during the maize life cycle. Furthermore, RNA-sequence-based gene expression profiling of PKs along a leaf developmental gradient and in mature bundle sheath and mesophyll cells indicated that ZmPK genes are involved in various physiological processes, such as cell-fate decisions, photosynthetic differentiation, and regulation of stomatal development. Our results provide new insights into the function and evolution of maize PKs and will be useful in studies aimed at revealing the global regulatory network of maize development, thereby contributing to the maize molecular breeding with enhanced quality traits.     

A BIL Population Derived from G. hirsutum and G. barbadense Provides a Resource for Cotton Genetics and Breeding

To provide a resource for cotton genetics and breeding, an interspecific hybridization between Gossypium hirsutum cv. Emian22 and G. barbadense acc. 3–79 was made. A population of 54 BILs (backcross inbred lines, BC₁F₈) was developed with the aim of transferring G. barbadense genes into G. hirsutum in order to genetically analyze these genes’ function in a G. hirsutum background and create new germplasms for breeding. Preliminary investigation of the morphological traits showed that the BILs had diverse variations in plant architecture, seed size, and fuzz color; the related traits of yield and fiber quality evaluated in 4 environments also showed abundant phenotypic variation. In order to explore the molecular diversity of the BIL population, 446 SSR markers selected at an average genetic distance of 10 cM from our interspecific linkage map were used to genotype the BIL population. A total of 393 polymorphic loci accounting for 84.4% MAF (major allele frequency) > 0.05 and 922 allele loci were detected, and the Shannon diversity index (I) was 0.417 per locus. The average introgression segment length was 16.24 cM, and an average of 29.53 segments were introgressed in each BIL line with an average background recovery of 79.8%. QTL mapping revealed 58 QTL associated with fiber quality and yield traits, and 47 favored alleles derived from the donor parent were discovered. This study demonstrated that the interspecific BIL population was enriched with much phenotypic and molecular variation which could be a resource for cotton genetics and breeding.     

Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens

Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained. The flavodoxin holoprotein is over-expressed in E. coli from the cloned gene using its endogenous promoter.     

Identification,molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens

The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching. The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.