Functional studies of a LIM -homeodomain transcription factor lmx1b in murine mid -hindbrain and limb development

This thesis is centered on applying molecular genetics to study pattern formation during animal development. More specifically, this thesis describes the functional studies of a LIM-homeodomain gene called lmx1b during murine embryogenesis. Lmx1b expression is restricted to the mid-hindbrain junction as well as to the dorsal mesenchyme of the limb, suggesting important functions during mid-hindbrain and limb development. To test these possibilities, lmx1b homozygous mutant mice were generated and their limb and CNS phenotypes examined. Lmx1b homozygous mutant mice exhibit a large reduction of mid-hindbrain structures, and that their limbs are symmetrical along the dorsal-ventral axis as the result of a dorsal to ventral transformation. Taken together, these studies define essential functions for lmx1b in mid-hindbrain patteming and in dorsal limb cell fate determination. However, the molecular mechanisms which accounts for these phenotypes are unknown, and whether lmx1b has same or distinctive functions during the mid-hindbrain and limb development is also unclear. ^ Recently, insight into molecular mechanisms of mid-hindbrain patterning and limb development has resulted from the identification of several factors with restricted expression patterns within these regions. These include the secreted factors wnt-1, fgf-8, wnt-7a and the transcription factors pax-2, and en-1. Targeted disruption of any of these genes in mice suggests that these genes might be involved in similar regulatory pathways. Analysis of the expression of these genes in lmx1b mutants demonstrates that lmxlb is not required for the initiation, but is required to maintain their expression at the mid-hindbrain junction. Thus, lmxlb is not required for specifying mid-hindbrain cell fates, rather, it functions to ensure the establishment or maintenance of a proper organizing center at the mid-hindbrain junction. Interestingly, lmxlb functions cell non-autonomously in chimera analysis, which indicates that lmx1b might regulate the expression of secreted factors such as wnt-1 and/or fgf-8 in the organizing center. In contrast, lmx1b functions cell autonomously in the dorsal limb to govern dorsal ventral limb development and its expression is regulated by with wnt-7a and en-1. However, single and double mutant analysis suggest that all three genes have partially overlapping functions as well as independent functions. The results point toward a complicated network of cross-talks among all three limb axes. ^

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

Insight into the strand exchange reaction promoted by the RecA protein of {\it Escherichia coli\/}

In vitro, RecA protein catalyses the exchange of single strands of DNA between different DNA molecules with sequence complementarity. In order to gain insight into this complex reaction and the roles of ATP binding and hydrolysis, two different approaches have been taken. The first is to use short single-stranded deoxyoligonucleotides as the ssDNA in strand exchange. These were used to determine the signal for hydrolysis and the structure of the RecA-DNA complex that hydrolyses ATP. I present a defined kinetic analysis of the nucleotide triphosphatase activity of RecA protein using short oligonucleotides as ssDNA cofactor. I compare the effects of both homopolymers and mixed base composition oligomers on the ATPase activity of RecA protein. I examine the steady state kinetic parameters of the ATPase reaction using these oligonucleotides as ssDNA cofactor, and show that although RecA can both bind to, and utilise, oligonucleotides 7 to 20 residues in length to support the repressor cleavage activity of RecA, these oligonucleotides are unable to efficiently stimulate the ATPase activity of RecA protein. I show that the K$\sb{\rm m}\sp{\rm ATP}$, the Hill coefficient for ATP binding, the extent of reaction, and k$\sb{\rm cat}$ are all a function of ssDNA chain length and that secondary structure may also play a role in determining the effects of a particular chain length on the ATPase activity of RecA protein.^ The second approach is to utilise one of the many mutants of RecA to gain insight into this complex reaction. The mutant selected was RecA1332. Surprisingly, in vitro, this mutant possesses a DNA-dependent ATPase activity. The K$\sb{\rm m}\sp{\rm ATP}$, Hill coefficient for ATP binding, and K$\sb{\rm m}\sp{\rm DNA}$ are similar to that of wild type. k$\sb{\rm cat}$ for the ATPase activity is reduced 3 to 12-fold, however. RecA1332 is unable to use deoxyoligonucleotides as DNA cofactors in the ATPase reaction, and demonstrates an increased sensitivity to inhibition by monovalent ions. It is able to perform strand exchange with ATP and ATP$\lbrack\gamma\rbrack$S but not with UTP, whereas the wild type protein is able to use all three nucleotide triphosphates. RecA1332 appears to be slowed in its ability to form intermediates and to convert these intermediates to products. (Abstract shortened by UMI.) ^

INDUCTION OF DNA STRAND BREAKS AND CROSS-LINKS BY THE NEW ANTICANCER AGENT 3,6-DIAZIRIDINYL-2,5-BIS(CARBOETHOXYAMINO) -1,4-BENZOQUINONE

The DNA breakage effect of the anticancer agent 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (AZQ, NSC-182986) on bacteriophage PM2 DNA was investigated using agarose gel electrophoresis. AZQ caused both single-stranded and double-stranded breaks after reduction with NaBH(,4), but it was not active in the native state. At 120 (mu)M, it degraded 50% of the closed circular form I DNA into 40% form II DNA (single-stranded break) and 10% form III DNA (double-stranded break). It produced a dose-response breakage between 1 (mu)M and 320 (mu)M. The DNA breakage exhibited a marked pH dependency. At 320 (mu)M, AZQ degraded 80% and 60% of form I DNA at pH 4 and 10 respectively, but none between pH 6 to 8. The DNA breakage at physiologic pH was greatly enhanced when 10 (mu)M cupric sulfate was included in the incubation mixture. The DNA strand scission was inhibited by catalase, glutathione, KI, histidine, Tiron, and DABCO. These results suggest that the DNA breakage may be caused by active oxygen metabolites including hydroxyl free radical. The bifunctional cross-linking activity of reduced AZQ on isolated calf thymus DNA was investigated by ethidium fluorescence assay. The cross-linking activity exhibited a similar pH dependency; highest in acidic and alkaline pH, inactive under neutral conditions. Using the alkaline elution method, we found that AZQ induced DNA single-stranded breaks in Chinese hamster ovary cells treated with 50 (mu)M of AZQ for 2 hr. The single-stranded break frequencies in rad equivalents were 17 with 50 (mu)M and 140 with 100 (mu)M of AZQ. In comparison, DNA cross-links appeared in cells treated with only 1 to 25 (mu)M of AZQ for 2 hr. The cross-linking frequencies in rad equivalents were 39 and 90 for 1 and 5 (mu)M of AZQ, respectively. Both DNA-DNA and DNa-protein cross-links were induced by AZQ in CHO cells as revealed by the proteinas K digestion assay. DNA cross-links increased within the first 4 hr of incubation in drug-free medium and slightly decreased by 12 hr, and most of the cross-links disappeared after cells were allowed to recovered for 24 hr.^ By electrochemical analysis, we found that AZQ was more readily reduced at acidic pH. However, incubation of AZQ with NaBH(,4) at pH 7.8 or 10, but not at 4, produced superoxide anion. The opening of the aziridinyl rings of AZQ at pH 4 was faster in the presence of NaBH(,4) than in its absence; no ring-opening was detected at pH 7.8 regardless of the inclusion of NaBH(,4). . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

DNA nucleotide excision repair gene single nucleotide polymorphisms and hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease caused by germline mutations in DNA mismatch repair(MMR) genes. The nucleotide excision repair(NER) pathway plays a very important role in cancer development. We systematically studied interactions between NER and MMR genes to identify NER gene single nucleotide polymorphism (SNP) risk factors that modify the effect of MMR mutations on risk for cancer in HNPCC. We analyzed data from polymorphisms in 10 NER genes that had been genotyped in HNPCC patients that carry MSH2 and MLH1 gene mutations. The influence of the NER gene SNPs on time to onset of colorectal cancer (CRC) was assessed using survival analysis and a semiparametric proportional hazard model. We found the median age of onset for CRC among MMR mutation carriers with the ERCC1 mutation was 3.9 years earlier than patients with wildtype ERCC1(median 47.7 vs 51.6, log-rank test p=0.035). The influence of Rad23B A249V SNP on age of onset of HNPCC is age dependent (likelihood ratio test p=0.0056). Interestingly, using the likelihood ratio test, we also found evidence of genetic interactions between the MMR gene mutations and SNPs in ERCC1 gene(C8092A) and XPG/ERCC5 gene(D1104H) with p-values of 0.004 and 0.042, respectively. An assessment using tree structured survival analysis (TSSA) showed distinct gene interactions in MLH1 mutation carriers and MSH2 mutation carriers. ERCC1 SNP genotypes greatly modified the age onset of HNPCC in MSH2 mutation carriers, while no effect was detected in MLH1 mutation carriers. Given the NER genes in this study play different roles in NER pathway, they may have distinct influences on the development of HNPCC. The findings of this study are very important for elucidation of the molecular mechanism of colon cancer development and for understanding why some mutation carriers of the MSH2 and MLH1 gene develop CRC early and others never develop CRC. Overall, the findings also have important implications for the development of early detection strategies and prevention as well as understanding the mechanism of colorectal carcinogenesis in HNPCC. ^

G870A polymorphism of cyclin D1 gene influences cancer risk - From epidemiological study to mechanism analysis

Alternate splicing of the cyclin D1 gene gives rise to transcript a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Through testing transcript a and transcript b in a series of human samples, we found that cyclin D1 transcript b is ubiquitously expressed as transcript a but in the lower abundance compared to transcript a. Epidemiological studies have reported that the cyclin D1 gene (CCND1) G870A polymorphism influences the risk for a variety of cancer. In this investigation, we examined the cyclin D1b levels in tumor samples with different genotypes and found that higher levels of cyclin D1b are expressed from the A allele than the G allele. Cyclin D1 is known as a cell cycle regulator facilitating the progression of the cell cycle from G1 to S phase in response to the mitogenic signals. It also interacts with several transcription factors and transcriptional coregulators to modulate their activities. It has been reported that cyclin D1a can substitute for estrogen to activate estrogen receptor α (ERα) mediated transcription and can induce the proliferation of estrogen responsive tissues. However the biological role of cyclin D1b in ERα transcriptional regulation has not been previously explored. In this study, we determined that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription. Cell proliferation assays provided the evidence that cyclin D1b negatively regulates estrogen responsive breast cancer cell growth. Taken together, our findings show that the CCND1 G870A polymorphism is correlated with increased levels of cyclin D1b and that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription providing evidence for the mechanism by which the CCND1 G870A polymorphism may be protective in certain types of breast cancer. ^

Single nucleotide polymorphisms (SNPs) associated with TGF-beta pathway and their significance in systemic sclerosis - A multilevel analysis

Systemic sclerosis (SSc) or Scleroderma is a complex disease and its etiopathogenesis remains unelucidated. Fibrosis in multiple organs is a key feature of SSc and studies have shown that transforming growth factor-β (TGF-β) pathway has a crucial role in fibrotic responses. For a complex disease such as SSc, expression quantitative trait loci (eQTL) analysis is a powerful tool for identifying genetic variations that affect expression of genes involved in this disease. In this study, a multilevel model is described to perform a multivariate eQTL for identifying genetic variation (SNPs) specifically associated with the expression of three members of TGF-β pathway, CTGF, SPARC and COL3A1. The uniqueness of this model is that all three genes were included in one model, rather than one gene being examined at a time. A protein might contribute to multiple pathways and this approach allows the identification of important genetic variations linked to multiple genes belonging to the same pathway. In this study, 29 SNPs were identified and 16 of them located in known genes. Exploring the roles of these genes in TGF-β regulation will help elucidate the etiology of SSc, which will in turn help to better manage this complex disease. ^

INFANT MORTALITY IN THE MEXICAN AMERICAN COMMUNITY: AN ANALYSIS OF DIFFERENTIALS IN INFANT, NEONATAL, AND POSTNEONATAL MORTALITY BY ETHNICITY AND PARENTAL NATIVITY, 1974-75 SINGLE LIVE BIRTH COHORT, HARRIS COUNTY, TEXAS

The paradoxically low infant mortality rates for Mexican Americans in Texas have been attributed to inaccuracies in vital registration and idiosyncracies in Mexican migration in rural areas along the U.S.-Mexico border. This study examined infant (IMR), neonatal (NMR), and postneonatal (PNMR) mortality rates of Mexican Americans in an urban, non-border setting, using linked birth and death records of the 1974-75 single live birth cohort (N = 68,584) in Harris County, Texas, which includes the city of Houston and is reported to have nearly complete birth and death registration. The use of parental nativity with the traditional Spanish surname criterion made it possible to distinguish infants of Mexican-born immigrants from those of Blacks, Anglos, other Hispanics, and later-generation, more Anglicized Mexican Americans. Mortality rates were analyzed by ethnicity, parental nativity, and cause of death, with respect to birth weight, birth order, maternal age, legitimacy status, and time of first prenatal care.^ While overall IMRs showed Spanish surname rates slightly higher than Anglo rates, infants of Mexican-born immigrants had much lower NMRs than did Anglos, even for moderately low birth weight infants. However, among infants under 1500 grams, presumably unable to be discharged home in the neonatal period, Mexican Americans had the highest NMR. The inconsistency suggested unreported deaths for Mexican American low birth weight infants after hospital discharge. The PNMR of infants of Mexican immigrants was also lower than for Anglos, and the usual mortality differentials were reversed: high-risk categories of high birth order, high maternal age, and late/no prenatal care had the lowest PNMRs. Since these groups' characteristics are congruent with those of low-income migrants, the data suggested the possibility of migration losses. Cause of death analysis suggested that prematurity and birth injuries are greater problems than heretofore recognized among Mexican Americans, and that home births and "shoebox burials" may be unrecorded even in an urban setting.^ Caution is advised in the interpretation of infant mortality rates for a Spanish surname population of Mexican origin, even in an urban, non-border area with reportedly excellent birth and death registration. ^