A trilocus sequence typing scheme for hospital epidemiology and subspecies differentiation of an important nosocomial pathogen, Enterococcus faecalis.

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST(M); based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.

Prognostic significance of xCT polymorphisms and expression in patients with advanced pancreatic cancer treated with chemotherapy

The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by pancreatic cancer cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione (GSH) levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including platinum. We examined 4 single nucleotide polymorphisms (SNPs) of the xCT gene in 269 advanced pancreatic cancer patients who received first line gemcitabine with or without cisplatin or oxaliplatin. Genotyping was performed using Taqman real-time PCR assays. A statistically significant correlation was noted between the 3' untranslated region (UTR) xCT SNP rs7674870 and overall survival (OS): Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in patients receiving gemcitabine in combination with platinum therapy (n = 145): MST was 10.5 versus 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). The 3' UTR xCT SNP rs7674870 may correlate with OS in pancreatic cancer patients receiving gemcitabine and platinum combination therapy. Paraffin-embedded core and surgical biopsy tumor specimens from 98 patients with metastatic pancreatic adenocarcinoma were analyzed by immunohistochemistry using an xCT specific antibody. xCT protein IHC expression scores were analyzed in relation to overall survival in 86 patients and genotype in 12 patients and no statistically significant association was found between the level of xCT IHC expression score and overall survival (p = 0.514). When xCT expression was analyzed in terms of treatment response, no statistically significant associations could be determined (p = 0.908). These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in pancreatic cancer.

Characterization of the beta-lactamase gene from {\it Enterococcus faecalis\/} strain HH22

The plasmid-encoded, constitutively produced $\beta$-lactamase gene from Enterococcus faecalis strain HH22 was genetically characterized. A restriction endonuclease map of the 5.1 kb EcoRI fragment encoding the enterococcal $\beta$-lactamase was prepared and compared with the restriction map of a cloned staphylococcal $\beta$-lactamase gene (from the naturally-occurring staphylococcal $\beta$-lactamase plasmid pI258). Comparison and hybridization studies showed that there were identical restriction sites in the region of the $\beta$-lactamase structural gene but not in the region surrounding this gene. Also the enterococcal $\beta$-lactamase plasmid did not encode resistance to mercury or cadmium which is encoded by the small, transducible staphylococcal $\beta$-lactamase plasmids. The nucleotide sequence of the enterococcal gene was shown to be identical to the published sequences of three of four staphylococcal type A $\beta$-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred-forty nucleotides upstream of the $\beta$-lactamase start codon were also determined for the inducible staphylococcal $\beta$-lactamase gene on pI258; this sequence was identical to that of the constitutively expressed enterococcal gene indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The gene for the enterococcal $\beta$-lactamase and an inducible staphylococcal $\beta$-lactamase were each cloned into a shuttle vector and then transformed into enterococcal and staphylococcal recipients. The major difference between the two host backgrounds was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene but no qualitative difference was seen between the two genera. Also a difference in the level of resistance to ampicillin was seen between the two backgrounds with the cloned enzymes by MIC and time-kill studies. The location of the enzyme was found to be host dependent since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell bound enzyme in the enterococcus. Based on the identity of the enterococcal $\beta$-lactamase and several staphylococcal $\beta$-lactamases, these data suggest recent spread of $\beta$-lactamase to enterococci and also suggest loss of a functional repressor. ^

H2B histone gene expression during {\it Xenopus laevis\/} development

Histone gene expression is replication-independent during oogenesis and early embryogenesis in amphibians; however, it becomes replication-dependent during later embryogenesis and remains replication-dependent through adulthood. In order to understand the mechanism for this switch in transcriptional regulation of histone gene expression during amphibian development, linker-scanning mutations were made in a Xenopus laevis H2B histone gene promoter by oligonucleotide site-directed mutagenesis and assayed by microinjection into oocytes and embryos. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CCAAT, and ATF motifs, required for maximal transcription in both oocytes and embryos. Factors binding to the CCAAT and ATF motifs are present in oocytes and embryos and increase slightly in abundance during early development. A sequence (CTTTACAT) in the frog H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is additionally required for maximal H2B transcription in frog embryos. Oocytes and embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer-binding proteins were isolated from Xenopus cDNA libraries by virtue of their similarity with the DNA binding (POU) domain of the ubiquitously expressed transcription factor Oct-1. The protein encoded by one of these genes, termed Oct-60, was localized mainly in the cytoplasm of oocytes and was also present in early embryos until the gastrula stage of development. Proteins encoded by the other two genes, Oct-25 and Oct-91, were present in embryos after the mid-blastula stage of development and decreased by early neurula stage. The activity of the Xenopus H2B octamer motif in embryos is not specifically associated with increased binding by Oct-1 or the appearance of novel octamer-binding proteins but requires the presence of an intact CCAAT motif. We found that synergistic interactions among promoter elements are important for full H2B promoter activity. The results suggest that transcription of the Xenopus H2B gene is replication-dependent when it is activated at the mid-blastula stage of development and that replication-dependent H2B transcription is mediated by Oct-1. ^

Transcriptional regulation and functional analysis of the Wilms' tumor gene WT1

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor which functions as a tumor suppressor. Defects in the WT1 gene can result in the development of nephroblastoma. WT1 is expressed during development, primarily in the metanephric kidney, the mesothelial lining of the abdomen and thorax, and the developing gonads. WT1 expression is tightly regulated and is essential for renal development. The WT1 gene encodes a protein with a proline-rich N-terminus which functions as a transcriptional repressor and C-terminus contains 4 zinc fingers that mediate DNA binding. WT1 represses transcription from a number of growth factors and growth factor receptors. WT1 mRNA undergoes alternative splicing at two sites, resulting in 4 mRNA species and polypeptide products. Exon 5, encoding 17 amino acids is alternatively spliced, and is located between the transcriptional repression domain and the DNA binding domain. The second alternative splice is the terminal 9 nucleotides of zinc finger 3, encoding the tripeptide Lys-Thr-Ser (KTS). The presence or absence of KTS within the zinc fingers of WT1 alters DNA binding.^ I have investigated transcriptional regulation of WT1, characterizing two means of repressing WT1 transcription. I have cloned a transcriptional silencer of the WT1 promoter which is located in the third intron of the WT1 gene. The silencer is 460 bp in length and contains an Alu repeat. The silencer functions in cells of non-renal origin.^ I have found that WT1 protein can autoregulate the WT1 promoter. Using the autoregulation of the WT1 promoter as a functional assay, I have defined differential consensus DNA binding motifs of WT1 isoforms lacking and containing the KTS tripeptide insertion. With these refined consensus DNA binding motifs, I have identified two additional targets of WT1 transcriptional repression, the proto-oncogenes bcl-2 and c-myc.^ I have investigated the ability of the alternatively spliced exon 5 to influence cell growth. In cell proliferation assays, isoforms of WT1 lacking exon 5 repress cell growth. WT1 isoforms containing exon 5 fail to repress cell growth to the same extent, but alter the morphology of the cells. These experiments demonstrate that the alternative splice isoforms of WT1 have differential effects on the function of WT1. These findings suggest a role for the alternative splicing of WT1 in metanephric development. ^

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

REGULATION OF THE EXPRESSION OF NAR OPERON ENCODING NITRATE REDUCTASE IN ESCHERICHIA COLI

The nar operon, which encodes the nitrate reductase in Escherichia coli, can be induced under anaerobic conditions without nitrate to a low level and with nitrate to a maximum level. The anaerobic formation of nitrate reductase is dependent upon the fnr gene product while the narL gene product is required for further induction by nitrate. The sequence was determined across the entire promoter and regulatory region of the nar operon. The translational start site of the first structural gene of the nar operon, narG gene, was established by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. The transcriptional start site and the level of the transcript was determined by S1 mapping procedure. One major transcript was identified which was initiated 50 base pair (bp) upstream from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, fully induced by nitrate anaerobically, and greatly reduced in a ${\rm Fnr\sp-}$ mutant. Deletions were created in the 5$\sp\prime$ nar regulatory sequence with either an intact nar operon or a nar::lacZ fusion. The expression of the plasmids with deletions were determined in a strain with wild type fnr and narL loci, a Fnr- mutant strain and a NarL- mutant strain. These experiments demonstrated that the $5\sp\prime$ limit of the nar operon lies at about $-210$ bp from the transcription start site. The region required for anaerobic induction by the fnr gene product is located around $-60$ bp. Two putative narL recognition sites were identified, one of which is around $-200$ and another immediately adjacent to the fnr recognition region. The deletion of the sequences around $-200$ rendered the remaining narL complex repressive and thus decreased the expression of nar operon, suggesting that the two potential narL sites interact with each other over a significant length of DNA. ^

The characterization of mouse carboxypeptidase N small subunit gene structure and presence in developing embryos

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits and two large subunits that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM. In this study, the mouse CPN small subunit (CPN1) coding region, gene structure, and chromosomal location were characterized and the expression of CPN1 was investigated in mouse embryos at different stages of development. The CPN1 gene, which was approximately 29 kb in length, contained nine exons and localized to mouse chromosome 19D2. The fifth and sixth exons of CPN1 encoded the amino acids necessary for substrate binding and catalytic activity. CPN1 RNA was expressed predominately in adult liver and contained a 1371 bp open reading frame encoding 457 amino acids. In the mouse embryo, CPN1 RNA was observed at 8.5 days post coitus (dpc), while its protein was detected at 10.5 dpc. In situ hybridization of the fetal liver detected CPN1 RNA in erythroid progenitor cells at 10.5, 13.5, and 16.5 dpc and in hepatocytes at 16.5 dpc. This was compared to the expression of the complement component C3, the parent molecule of complement anaphylatoxin C3a. Consistently throughout the experiments, CPN1 message and protein preceded the expression of C3. To obtain a better understanding of the biological significance of CPN1 in vivo, studies were initiated to produce a genetically engineered mouse in which the CPN1 gene was ablated. To facilitate this project a targeting vector was constructed by removing the functionally important fifth and sixth exons of the CPN1 gene. Collectively, these studies have: (1) provided important detailed information regarding the structure and organization of the murine CPN1 gene, (2) yielded insights into the developmental expression of mouse CPN1 in relationship to C3 expression, and (3) set the stage for the generation of a CPN1 “knock-out” mouse, which can be used to determine the biological significance of CPN1 in both normal and diseased conditions. ^

An extracytoplasmic function sigma factor operon regulates Myxococcus xanthus developmental gene expression

Myxococcus xanthus is a Gram-negative soil bacterium that undergoes multicellular development when high-density cells are starved on a solid surface. Expression of the 4445 gene, predicted to encode a periplasmic protein, commences 1.5 h after the initiation of development and requires starvation and high density conditions. Addition of crude or boiled supernatant from starving high-density cells restored 4445 expression to starving low-density cells. Addition of L-threonine or L-isoleucine to starving low-density cells also restored 4445 expression, indicating that the high-density signaling activity present in the supernatant might be composed of extracellular amino acids or small peptides. To investigate the circuitry integrating these starvation and high-density signals, the cis- and trans-acting elements controlling 4445 expression were identified. The 4445 transcription start site was determined by primer extension analysis to be 58 by upstream of the predicted translation start site. The promoter region contained a consensus sequence characteristic of e&barbelow;xtrac&barbelow;ytoplasmic f&barbelow;unction (ECF) sigma factor-dependent promoters, suggesting that 4445 expression might be regulated by an ECF sigma factor-dependent pathway, which are known to respond to envelope stresses. The small size of the minimum regulatory region, identified by 5′-end deletion analysis as being only 66 by upstream of the transcription start site, suggests that RNA polymerase could be the sole direct regulator of 4445 expression. To identify trans-acting negative regulators of 4445 expression, a strain containing a 4445-lacZ was mutagenized using the Himar1-tet transposon. The four transposon insertions characterized mapped to an operon encoding a putative ECF sigma factor, ecfA; an anti-sigma factor, reaA; and a negative regulator, reaB. The reaA and the reaB mutants expressed 4445 during growth and development at levels almost 100-fold higher than wild type, indicating that these genes encode negative regulators. The ecfA mutant expressed 4445-lacZ at basal levels, indicating that ecfA is a positive regulator. High Mg2+ concentrations over-stimulated this ecfA pathway possibly due to the depletion of exopolysaccharides and assembled type IV pili. These data indicate that the ecfA operon encodes a new regulatory stress pathway that integrates and transduces starvation and cell density cues during early development and is also responsive to cell-surface alterations.^

Candidate gene identification following linkage: Search for hypertension susceptibility genes

Following up genetic linkage studies to identify the underlying susceptibility gene(s) for complex disease traits is an arduous yet biologically and clinically important task. Complex traits, such as hypertension, are considered polygenic with many genes influencing risk, each with small effects. Chromosome 2 has been consistently identified as a genomic region with genetic linkage evidence suggesting that one or more loci contribute to blood pressure levels and hypertension status. Using combined positional candidate gene methods, the Family Blood Pressure Program has concentrated efforts in investigating this region of chromosome 2 in an effort to identify underlying candidate hypertension susceptibility gene(s). Initial informatics efforts identified the boundaries of the region and the known genes within it. A total of 82 polymorphic sites in eight positional candidate genes were genotyped in a large hypothesis-generating sample consisting of 1640 African Americans, 1339 whites, and 1616 Mexican Americans. To adjust for multiple comparisons, resampling-based false discovery adjustment was applied, extending traditional resampling methods to sibship samples. Following this adjustment for multiple comparisons, SLC4A5, a sodium bicarbonate transporter, was identified as a primary candidate gene for hypertension. Polymorphisms in SLC4A5 were subsequently genotyped and analyzed for validation in two populations of African Americans (N = 461; N = 778) and two of whites (N = 550; N = 967). Again, SNPs within SLC4A5 were significantly associated with blood pressure levels and hypertension status. While not identifying a single causal DNA sequence variation that is significantly associated with blood pressure levels and hypertension status across all samples, the results further implicate SLC4A5 as a candidate hypertension susceptibility gene, validating previous evidence for one or more genes on chromosome 2 that influence hypertension related phenotypes in the population-at-large. The methodology and results reported provide a case study of one approach for following up the results of genetic linkage analyses to identify genes influencing complex traits. ^

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

Gene clustering of the small leucine -rich repeat gene family

The small leucine-rich repeat proteoglycans (or SLRPs) are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins. The LRR is a protein folding motif composed of 20–30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization. In mammals, the SLRPs are abundant in connective tissues, such as bones, cartilage, tendons, skin, and blood vessels. We have discovered a new member of the class I small leucine rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N-terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9g21.3 where asporin is part of a SLRP gene cluster that includes ECM2, osteoadherin, and osteoglycin. This gene cluster of four LRR-encoding genes is embedded in a 238 kilobase intron of another novel gene named Tes9orf that is expressed primarily in the testes of the adult mouse. The SLRP genes are not present in Drosophila or C. elegans , but reside in three separate gene clusters in the puffer fish, mice and humans. Targeted disruption of individual mouse SLRP genes display minor connective tissue defects such as skin fragility, tendon laxity, minor growth plate defects, and mild osteoporosis. However, double and triple knockouts of SLRP genes exacerbate these phenotypes. Both the double epiphycan/biglycan and the triple PRELP/fibromodulin/biglycan knockout mice exhibit premature osteoarthritis. ^