A molecular study of the rhodopsin locus in patients with retinitis pigmentosa

DNA for this study was collected from a sample of 133 retinitis pigmentosa (RP) patients and the rhodopsin locus molecularly analyzed by linkage and for disease specific mutations. The cohort of patients consisted of 85 individuals diagnosed with autosomal dominant RP (adRP), and 48 patients representing other forms of retinitis pigmentosa or retinal dystrophy related disease. In three large families with adRP rhodopsin was excluded from linkage to the disease locus. A search for subtle mutations in the rhodopsin coding region using single strand conformational polymorphisms (SSCP) and sequencing detected a total of 14 unique sequence variants in 24 unrelated patients. These variants included one splicing variant, 5168 -1G-A, one deletion variant of 17 base pairs causing a frame shift at codon 332, and 12 misense variants: Pro23His, Leu46Arg, Gly106Trp, Arg135Pro, Pro171Glu, Pro180Ala, Glu181Lys, Asp190Asn, His211Arg, Ser270Arg, Leu328Pro and Pro347Thr. All but three of the missense variants change amino acids that are evolutionarily conserved. The Pro23His mutation was found in 10 unrelated individuals with family histories of adRP and not in any normal controls (over 80 chromosomes tested). The Pro180Ala mutation was present in a patient with simplex RP and probably represents a new mutation. Three normal polymorphic nucleotide substitutions, A-269-G, T-3982-C, and G-5145-A, were also identified. We conclude, based on this study, that 25% of adRP cases are attributable to rhodopsin mutations.^ Clinical data, including ERG results and visual field testing, was available for patients with eleven different mutations. The eleven patients were all diagnosed with RP, however the severity of the disease varied with five patients mildly affected and diagnosed with type II adRP and 5 patients severely affected and diagnosed with type I adRP. The patient with simplex RP was mildly affected. The location of the mutations within the rhodopsin protein was randomly associated with the severity of the disease in those patients evaluated. However, four mutations, Pro23His, Leu46Arg, Pro347Thr, and 5168 -1G-A, are particularly interesting. The Pro23His mutation appears to have radiated from a recent common ancestor of the affected patients as all of them share a common haplotype at the rhodopsin locus. The Leu46Arg mutation causes an unusually severe form of RP. Hydropathy analysis of the mutated sequence revealed a marked change in the hydrophobicity of this first transmembrane spanning region. Codon 347 has been the target of multiple mutations with at least six documented changes at the position, significantly more than expected by a random distribution of mutations. Finally the splice-site variant is extremely variable in its expression in the family studied. Similar mutations have been reported in other cases of adRP and postulated to be involved in autosomal recessive RP (arRP). Mechanisms to account for the variable expression of rhodopsin mutations in relation to RP heterogeneity are discussed. (Abstract shortened by UMI.) ^

Functional analysis of anionic phospholipids in cell metabolism in {\it Escherichia coli\/}

The major goal of this work was to understand the function of anionic phospholipid in E. coli cell metabolism. One important finding from this work is the requirement of anionic phospholipid for the DnaA protein-dependent initiation of DNA replication. An rnhA mutation, which bypasses the need for the DnaA protein through induction of constitutive stable DNA replication, suppressed the growth arrest phenotype of a $pgsA$ mutant in which the synthesis of anionic phospholipid was blocked. The maintenance of plasmids dependent on an $oriC$ site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein. In addition, structural and functional requirements of two major anionic phospholipids, phosphatidylglycerol and cardiolipin, were examined. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for the naturally occurring phosphatidylglycerol. The requirement for phosphatidylglycerol was concluded to be more than maintenance of the proper membrane surface charge. Examination of the role of cardiolipin revealed its ability to replace the zwitterionic phospholipid, phosphatidylethanolamine, in maintaining an optimal membrane lipid organization. This work also reported the DNA sequence of the cls gene, which encodes the CL synthase responsible for the synthesis of cardiolipin. ^

The biology and mechanism of chemoresistance of brain metastases

Primary brain neoplasms and metastases to the brain are generally resistant to systemic chemotherapy. The purpose of theses studies was to determine the mechanism(s) for this resistance. We have developed a model to study the biology of brain metastasis by injecting metastatic K1735 melanoma cells into the carotid artery of syngeneic C3H/HeN or nude mice. The resulting brain lesions are produced in the parenchyma of the brain. Mice with subcutaneous or brain melanoma lesions were treated intravenously with doxorubicin (DXR) (7 mg/kg). The s.c. lesions regressed in most of the mice whereas no therapeutic benefits were produced in mice with brain metastases. The intravenous injection of sodium fluorescine revealed that the blood-brain barrier (BBB) is intact in and around brain metastases smaller than 0.2 mm$\sp2$ but not in larger lesions, implying that the BBB is not a major obstacle for chemotherapy of brain metastases.^ Western blot and FACS analyses revealed that K1735 melanoma brain metastases expressed high levels of P-glycoprotein (P-gp) as compared to s.c. tumors or in vitro cultures. Similarly, K1735 cells from brain metastases expressed higher levels of mdrl mRNA. This increased expression of mdrl was due to adaptation to the local brain environment. We base this conclusion on the results of two studies. First, K1735 cells from brain metastases cultured for 7 days lost the high mdrl expression. Second, in crossover experiments K1735 cells from s.c. tumors (low mdrl expression) implanted into the brain exhibited high levels of mdrl expression whereas cells from brain metastases implanted s.c. lost the high level mdrl expression.^ To investigate the mechanism by which the brain environment upregulates mdrl expression of the K1735 cells we first studied the regulation of P-gp in brain endothelial cells. Since astrocytes are closely linked with the BBB we cocultured brain endothelial cells for 3 days with astrocytes. These endothelial cells expressed high levels of mdrl mRNA and protein whereas endothelial cells cocultured with endothelial cells or fibroblasts did not. We next cocultured K1735 melanoma cells with astrocytes. Here again, astrocytes (but not fibroblasts or tumor cells) uprelated the mdrl expression in K1735 tumor cells. This upregulation inversely correlated with intracellular drug accumulation and sensitivity to DXR.^ The data conclude that the resistance of melanoma brain metastases to chemotherapy is not due to an intact BBB but to the upregulation of the mdrl gene by the organ microenvironment, i.e., the astrocytes. This epigenetic mediated resistance to chemotherapy has wide implications for the therapy of brain metastases. ^

Molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$-adrenergic receptor

The $\beta$-adrenergic receptor ($\beta$AR), which couples to G$\sb{\rm s}$ and activates adenylylcyclase, has been a prototype for studying the activation and desensitization of G-protein-coupled receptors. The main objective of the present study is to elucidate the molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$AR.^ Activation of cAPK or PKC causes a rapid desensitization of $\beta$AR stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the $\beta$AR, RRSSK$\sp{263}$. To determine the role of the individual serines in the cAPK- and PKC-meditated desensitizations, wild type (WT) and mutant $\beta$ARs containing the substitutions, Ser$\sp{261} \to$ A, Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and Ser$\sp{261/262} \to$ A, were constructed and stably transfected into L cells. The cAPK-mediated desensitization was decreased 70-80% by the Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and the Ser$\sp{261/262} \to$ A mutations, but was not altered by the Ser$\sp{261} \to$ A substitution, demonstrating that Ser$\sp{262}$ was the primary site of the cAPK-induced desensitization. The PMA/PKC-induced desensitization was unaffected by either of the single serine to alanine substitutions, but was reduced 80% by the double serine to alanine substitution, suggesting that either serine was sufficient to confer the PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser$\sp{262} \to$ A, was partially uncoupled. The Ser$\sp{262} \to$ D mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor.^ To accomplish the in vivo phosphorylation of the $\beta$AR, we used two epitope-modified $\beta$ARs, hemagglutinin-tagged $\beta$AR (HA-$\beta$AR) and 6 histidine-tagged $\beta$AR (6His-$\beta$AR), for a high efficiency purification of the $\beta$AR. Neither HA-$\beta$AR nor 6His-$\beta$AR altered activation and desensitization of the $\beta$AR significantly as compared to unmodified wild type $\beta$AR. 61% recovery of ICYP-labeled $\beta$AR was obtained with Ni-NTA column chromatography.^ The truncation 354 mutant $\beta$AR(T354), lacking putative $\beta$ARK site(s), displayed a normal epinephrine stimulation of adenylylcyclase. Although 1.0 $\mu$M epinephrine induced 60% less desensitization in T354 as compared to wild type $\beta$AR, 1.0 $\mu$M epinephrine-mediated desensitization in T354 was 35% greater than PGE$\sb1$-mediated desensitization, which is essentially identical in both WT and T354. These results suggested that sequences downstream of residue 354 may play a role in homologous desensitization and that internalization may be attributed to the additional desensitization besides the cAMP mechanism in T354 $\beta$AR. (Abstract shortened by UMI.) ^

Molecular mechanisms of prostatic carcinoma growth and progression

Prostate cancer represents the most commonly diagnosed malignancies in American men and is the second leading cause of male cancer deaths. The overall objectives of this research were designed to understand the cellular and molecular mechanisms of prostatic carcinoma growth and progression. This dissertation was divided into two major parts: (1) to clone and characterize soluble factor(s) associated with bone that may mediate prostatic carcinoma growth and progression; (2) to investigate the roles of extracellular matrix in prostatic carcinogenesis.^ The propensity of prostate cancer cells to metastasize to the axial skeleton and the subsequent osteoblastic reactions observed in the bone indicate the possible reciprocal cellular interaction between prostate cancer cells and the bone microenvironment. To understand the molecular and cellular basis of this interaction, I focused on the identification and cloning of soluble factor(s) from bone stromal cells that may exert direct mitogenic action on cultured prostate cells. A novel BPGF-1 gene expressed specifically by bone and male accessory sex organs (prostate, seminal vesicles, and coagulating gland) was identified and cloned.^ The BPGF-1 was identified and cloned from a cDNA expression library prepared from a human bone stromal cell line, MS. The conditioned medium (CM) of this cell line contains mitogenic materials that induce human prostate cancer cell growth both in vivo and in vitro. The cDNA expression library was screened by an antibody prepared against the mitogenic fraction of the CM.^ The cloned BPGF-1 cDNA comprises 3171 nucleotides with a single open reading frame of 1620 nucleotides encoding 540 amino acids. The BPGF-1 gene encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity in human cells and tissues, but only one transcript (2.5 kb) in rat and mouse tissues. Southern blot analysis of human genomic DNA revealed a single BPGF-1 gene. The BPGF-1 gene is expressed predominantly in bone and seminal vesicles, but at a substantially lower level in prostate. Polyclonal antibodies generated from synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a putative BPGF-1 protein with an apparent molecular weight of 70 kDa. The conditioned media isolated from COS cells transfected with BPGF-1 cDNA stimulated the proliferation and increased the anchorage-independent growth of prostate epithelial cells. These findings led us to hypothesize that BPGF-1 expression in relevant organs, such as prostate, seminal vesicles, and bone, may lead to local prostate cancer growth, metastasis to the seminal vesicles, and subsequently dissemination to the skeleton.^ To assess the importance of extracellular matrix in prostatic carcinogenesis, the role of extracellular matrix in induction of rat prostatic carcinoma growth in vivo was evaluated. NbE-1, a nontumorigenic rat prostatic epithelial cell line, was induced to form carcinoma in athymic nude hosts by coinjecting them with Matrigel and selected extracellular matrix components. Induction of prostatic tumor formation by laminin and collagen IV was inhibited by their respective antibodies. Prostatic epithelial cells cloned from the tumor tissues were found to form tumors in athymic nude hosts in the absence of exogenously added extracellular matrix. These results suggest that extracellular matrix induce irreversibly prostatic epithelial cells that behave distinctively different from the parental prostatic epithelial cell line. ^

The molecular and genetic characterization of the MADS box transcription factor {\it Drosophila\/} MEF2

The myocyte enhancer factor (MEF)-2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates, but the precise position of these regulators within the genetic hierarchy leading to myogenesis is unclear. The MEF2 proteins bind to a conserved A/T-rich DNA sequence present in numerous muscle-specific genes, and they are expressed in the cells of the developing somites and in the embryonic heart at the onset of muscle formation in mammals. The MEF2 genes belong to the MADS box family of transcription factors, which control specific programs of gene expression in species ranging from yeast to humans. Each MEF2 family member contains two highly conserved protein motifs, the MADS domain and the MEF2-specific domain, which together provide the MEF2 factors with their unique DNA binding and dimerization properties. In an effort to further define the function of the MEF2 proteins, and to evaluate the degree of conservation shared among these factors and the phylogenetic pathways that they regulate, we sought to identify MEF2 family members in other species. In Drosophila, a homolog of the vertebrate MEF2 genes was identified and termed D-mef2. The D-MEF2 protein binds to the consensus MEF2 element and can activate transcription through tandem copies of that site. During Drosophila embryogenesis, D-MEF2 is specific to the mesoderm germ layer of the developing embryo and becomes expressed in all muscle cell types within the embryo. The role of D-mef2 in Drosophila embryogenesis was examined by generating a loss-of-function mutation in the D-mef2 gene. In embryos homozygous for this mutant allele, somatic, cardiac, and visceral muscles fail to differentiate, but precursors of these myogenic lineages are normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2 and suggest that this program has been conserved from Drosophila to mammals. ^

Molecular analysis of the human $\gamma$-chain T cell receptor genes

In our studies we have focused on the issue of variability and diversity of the $\gamma$ (or $\delta)$ chain T cell receptor (TCR) genes by studying cDNA transcripts in peripheral blood mononuclear cells or $\gamma\delta$ TCR+ T cell clones. The significance of these studies lies in the better understanding of the molecular biology of the $\gamma\delta$ T cell receptor as well as in answering the question whether certain molecular forms predominate in $\gamma\delta$ T cells exhibiting specific immunologic functions. We establish that certain $\gamma$-chain TCR genes exhibit particular patterns of rearrangements in cDNA transcripts in normal individuals. V$\gamma$I subgroup were shown to preferentially rearrange to J$\gamma$2C$\gamma$2 gene segments. These preferential VJC rearrangements, may have implications regarding the potential for diversity and polymorphism of the $\gamma$-chain TCR gene. In addition, the preferential association of V$\gamma$I genes with J$\gamma$2C$\gamma$2, which encode a non-disulfide-linked $\gamma\delta$ TCR, suggests that $\gamma$ chains utilizing V$\gamma$I are predominantly expressed as non-disulfide-linked $\gamma\delta$ TCR heterodimers. The implications of this type of expression remain to be determined. We identified two alternative splicing events of the $\gamma$-chain TCR genes occurring in high frequency in all the normal individuals examined. These events may suggest additional mechanisms of regulation and control as well as diversification of $\gamma\delta$ TCR gene expression. The question whether particular forms of $\gamma$ or $\delta$-chain TCR genes are involved in HLA Class I recognition by specific $\gamma\delta$ cytotoxic T cell clones was addressed. Our results indicated that the T cell clones expressed identical $\gamma$ but distinct $\delta$-chains suggesting that the specificity for recognition of HLA-A2 or HLA-A3 may be conferred by the $\delta$-chain TCR. The issue of the degree of diversity and polymorphism of the $\delta$-chain TCR genes in a patient with a primary immunodeficiency (Omenn's syndrome) was addressed. A limited pattern of rearrangements in peripheral blood transcripts was found, suggesting that a limited $\gamma\delta$ TCR repertoire may be expressed in this particular primary immunodeficiency syndrome. Overall, our findings suggest that $\delta$-chain TCR genes exhibit the potential for significant diversity and that there are certain preferential patterns of expression that may be associated with particular immunologic functions. ^

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Regulation of the expression of {\it mutS, mutL}, and {\it mutH\/} mismatch repair genes in {\it Escherichia coli\/} K-12

Post-replication DNA mismatch repair plays crucial roles in mutation avoidance and maintenance of chromosome stability in both prokaryotes and eukaryotes. In humans, deficiency in this repair system leads to a predisposition for certain cancers. The biochemistry of this repair system has been best studied in a model bacterium Escherichia coli. In this thesis, regulation of expression of mutS, mutL and mutH genes, whose products mediate methyl-directed mismatch (MDM) repair in E. coli, is investigated. One-step affinity purification schemes were developed to purify E. coli MutS, MutL and MutH proteins fused to a His-6-affinity tag. His-6-MutS exhibited the same mismatch binding activity and specificity as the native MutS protein. Purified His-6-MutS, -MutL and -MutH proteins were used to develop quantitative Western blotting assays for amounts of MutS, MuL and MutH proteins under various conditions. It was found that the three proteins were present in relatively low amounts in exponentially growing cells and MutS and MutH were diminished in stationary-phase cells. Further studies indicated that the drop in the amounts of MutS and MutH proteins in stationary-phase cells was mediated through RpoS, a key global regulator of stationary-phase transition. In both exponential- and stationary-phase cells, MutS amount was also negatively regulated by the Hfq (HF-I) global regulator, which is required for RpoS translation, through an RpoS-independent mechanism. $\beta$-galactosidase assays of mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally, and RNase T2 protection assays revealed that Hfq destabilizes mutS transcripts in exponentially growing cells. To study the relation between regulation of MDM repair and mutagenesis, amounts of MutS, MutL and MutH were measured in starved cells undergoing adaptive mutagenesis. It was found that MutS amount dropped drastically, MutH amount dropped slightly, whereas MutL amount remained essentially constant in starved cells. Overexpression of MutL did not reverse the drop in the amounts of MutS or MutH protein. These results ruled out several explanations for a phenomenon in which overexpression of MutL, but not MutS, reversed adaptive mutagenesis. The findings further suggested that functional MutL is limiting during adaptive mutagenesis. The implications of regulation of the MDM repair are discussed in the context of mutagenesis, pathogenesis and tumorigenesis. ^

Regulatory factors and control of anthrax toxin gene expression

Bacillus anthracis plasmid pXO1 carries genes for three anthrax toxin proteins, pag (protective antigen), cya (edema factor), and lef (lethal factor). Expression of the toxin genes is enhanced by two signals: CO$\sb2$/bicarbonate and temperature. The CO$\sb2$/bicarbonate effect requires the presence of pXO1. I hypothesized that pXO1 harbors a trans-acting regulatory gene(s) required for CO$\sb2$/bicarbonate-enhanced expression of the toxin genes. Characterization of such a gene(s) will lead to increased understanding of the mechanisms by which B. anthracis senses and responds to host environments.^ A regulatory gene (atxA) on pXO1 was identified. Transcription of all three toxin genes is decreased in an atxA-null mutant. There are two transcriptional start sites for pag. Transcription from the major site, P1, is enhanced in elevated CO$\sb2$. Only P1 transcripts are significantly decreased in the atxA mutant. Deletion analysis of the pag upstream region indicates that the 111-bp region upstream of the P1 site is sufficient for atxA-mediated increase of this transcript. The cya and lef genes each have one apparent transcriptional start site. The cya and lef transcripts are significantly decreased in the atxA mutant. The atxA mutant is avirulent in mice. The antibody response to all three toxin proteins is significantly decreased in atxA mutant-infected mice. These data suggest that the atxA gene product activates expression of the toxin genes and is essential for virulence.^ Since expression of the toxin genes is dependent on atxA, whether increased toxin gene expression in response to CO$\sb2$/bicarbonate and temperature is associated with increased atxA expression was investigated. I monitored steady state levels of atxA mRNA and AtxA protein in different growth conditions. The results indicate that expression of atxA is not influenced by CO$\sb2$/bicarbonate. Steady state levels of atxA mRNA and AtxA protein are higher at 37$\sp\circ$C than 28$\sp\circ$C. However, increased pag expression at high temperature can not be attributed directly to increased atxA expression.^ There is evidence that an additional factor(s) may be involved in regulation of pag. Expression of pag in strains overproducing AtxA is significantly decreased compared to the wildtype strain. A specific interaction of tagged-AtxA with the pag upstream DNA has not been demonstrated. Furthermore, four proteins in B. anthracis extract can be co-immunoprecipitated with tagged-AtxA. Amino-terminal sequence of one protein has been determined and found highly homologous to chaperonins of GroEL family. Studies are under way to determine if this GroEL-like protein interactions with AtxA and plays any role in atxA-mediated activation of toxin genes. ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

Antigenic variation in Lyme disease spirochetes by segmental recombination of VMP-like sequence cassettes

Lyme disease is a multisystemic disorder caused by tick-borne infection of humans or other mammalian hosts with Borrelia burgdorferi. If untreated, the spirochetes can persist in the mammalian host for months or years. The mechanisms by which Lyme disease spirochetes evade the immune response have not been determined. In this study, we have identified and characterized an elaborate genetic system in the Lyme disease spirochete B. burgdorferi that promotes extensive antigenic variation of a 34-kDa surface-exposed lipoprotein, VlsE. A 28-kilobase linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. The presence of lp28-1 correlates with the high-infectivity phenotype in B. burgdorferi strains tested. Segments of the 15 non-expressed (silent) vls cassette sequences located upstream of vlsE are able to recombine into the centra vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. When compared to parental VlsE, VlsE variants progressively accumulate sequence changes during the period of 4, 7, 14, 21, and 28 days post infection in C3H/HeN mice. However, no recombination was detected during the period of 28-day in vitro culture, suggesting in vivo induction of VlsE antigenic variation. Adaptive immune responses do not appear to play a significant role in this induction, since similar recombination events were also observed in immunodeficient SCID mice. The $5\sp\prime$ and $3\sp\prime$ noncassette regions of vlsE are apparently not subject to recombination and sequence variation. The structure and sequence of the silent vls cassette locus is preserved during the process of the VlsE antigenic variation, consistent with a nonreciprocal recombination mechanism. This combinatorial form of antigenic variation could potentially yield millions of VlsE variants in the mammalian host, and thereby contribute to immune evasion, long-term survival, and pathogenesis of B. burgdorferi. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

The association of genetic groups 1, 2, and 3 of Mycobacterium tuberculosis with pulmonary and extra-pulmonary tuberculosis

Objective. To investigate the association of the three major genetic groups of Mycobacterium tuberculosis with pulmonary and extra-pulmonary tuberculosis in clustered and non-clustered TB cases in the Houston area. ^ Study design. Secondary analysis of an ambi-directional study. ^ Study population. Three hundred fifty-eight confirmed cases of tuberculosis in the Houston that occurred between October 1995 and May 1997, who had been interviewed by the Houston T13 Initiative staff at Baylor College of Medicine, and whose isolates have had their DNA fingerprint and genetic group determined. ^ Exclusions. Individuals whose mycobacterial genotype was unknown, or whose data variables were unavailable. ^ Source of data. Laboratory results, patient interviews, and medical records at clinics and hospitals of the study population. ^ Results. In clustered cases, the majority of both, pulmonary and extra-pulmonary TB cases were caused by genetic group 1. Independent factors were assessed to determine the interactions that may influence the site of infection or increase the risk for one site or another. HIV negative males were protected against extra-pulmonary TB compared to HIV negative females. Individuals ages 1–14 years were at higher risk of having extra-pulmonary TB. Group 3 organisms were found less frequently in the total population in general, especially in extra-pulmonary disease. This supports the evidence in previous studies that this group is the least virulent and genetically distinct from the other two groups. Group 1 was found more frequently among African Americans than other ethnic groups, a trend for future investigations. ^ Among the non-clustered cases, group 2 organisms were the majority of the organisms found in both sites. They were also the majority of organisms found in African Americans, Caucasians, and Hispanics causing the majority of the infections at both sites. However, group 1 organisms were the overwhelming majority found in Asian/Pacific Islander individuals, which may indicate these organisms are either endemic to that area, or that there is an ethnic biological factor involved. This may also be due to a systematic bias, since isolates from individuals from that geographic region lack adequate copies of the insertion sequence IS6110, which leads to their placement in the non-clustered population. ^ The three genetic groups of Mycobacterium tuberculosis were not found equally distributed between sites of infection in both clustered and non-clustered cases. Furthermore, these groups were not distributed in the same patterns among the clustered and non-clustered cases, but rather in distinct patterns. ^