Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins.

Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37 degrees C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C RTR1 (regulator of transcription). The core RNAPII subunits RPB5, RPB7, and RPB9 were isolated as potent high-copy-number suppressors of the rtr1Delta temperature-sensitive growth phenotype, and deletion of the nonessential subunits RPB4 and RPB9 hypersensitized cells to RTR1 overexpression. Disruption of RTR1 resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently, rtr1Delta cells are defective in inducible transcription from the GAL1 promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.

The sequence comparison index: A novel method for comparing proteins and proteomes

Historically morphological features were used as the primary means to classify organisms. However, the age of molecular genetics has allowed us to approach this field from the perspective of the organism's genetic code. Early work used highly conserved sequences, such as ribosomal RNA. The increasing number of complete genomes in the public data repositories provides the opportunity to look not only at a single gene, but at organisms' entire parts list. ^ Here the Sequence Comparison Index (SCI) and the Organism Comparison Index (OCI), algorithms and methods to compare proteins and proteomes, are presented. The complete proteomes of 104 sequenced organisms were compared. Over 280 million full Smith-Waterman alignments were performed on sequence pairs which had a reasonable expectation of being related. From these alignments a whole proteome phylogenetic tree was constructed. This method was also used to compare the small subunit (SSU) rRNA from each organism and a tree constructed from these results. The SSU rRNA tree by the SCI/OCI method looks very much like accepted SSU rRNA trees from sources such as the Ribosomal Database Project, thus validating the method. The SCI/OCI proteome tree showed a number of small but significant differences when compared to the SSU rRNA tree and proteome trees constructed by other methods. Horizontal gene transfer does not appear to affect the SCI/OCI trees until the transferred genes make up a large portion of the proteome. ^ As part of this work, the Database of Related Local Alignments (DaRLA) was created and contains over 81 million rows of sequence alignment information. DaRLA, while primarily used to build the whole proteome trees, can also be applied shared gene content analysis, gene order analysis, and creating individual protein trees. ^ Finally, the standard BLAST method for analyzing shared gene content was compared to the SCI method using 4 spirochetes. The SCI system performed flawlessly, finding all proteins from one organism against itself and finding all the ribosomal proteins between organisms. The BLAST system missed some proteins from its respective organism and failed to detect small ribosomal proteins between organisms. ^

Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia.

A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin's activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1 locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow-derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types.

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina.

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

Cloning and characterization of the full length cDNA and promoter of mouse tissue transglutaminase

Transglutaminases are a family of enzymes that catalyze the covalent cross-linking of proteins through the formation of $\varepsilon$-($\gamma$-glutaminyl)-lysyl isopeptide bonds. Tissue transglutaminase (Tgase) is an intracellular enzyme which is expressed in terminally differentiated and senescent cells and also in cells undergoing apoptotic cell death. To characterize this enzyme and examine its relationship with other members of the transglutaminase family, cDNAs, the first two exons of the gene and 2 kb of the 5$\sp\prime$ flanking region, including the promoter, were isolated. The full length Tgase transcript consists of 66 bp of 5$\sp\prime$-UTR (untranslated) sequence, an open reading frame which encodes 686 amino acids and 1400 bp of 3$\sp\prime$-UTR sequence. Alignment of the deduced Tgase protein sequence with that of other transglutaminases revealed regions of strong homology, particularly in the active site region.^ The Tgase cDNA was used to isolate and characterize a genomic clone encompassing the 5$\sp\prime$ end of the mouse Tgase gene. The transcription start site was defined using genomic and cDNA clones coupled with S1 protection analysis and anchored PCR. This clone includes 2.3 kb upstream of the transcription start site and two exons that contain the first 256 nucleotides of the mouse Tgase cDNA sequence. The exon intron boundaries have been mapped and compared with the exon intron boundaries of three members of the transglutaminase family: human factor XIIIa, the human keratinocyte transglutaminase and human erythrocyte band 4.1. Tissue Tgase exon II is similar to comparable exons of these genes. However, exon I bears no resemblance with any of the other transglutaminase amino terminus exons.^ Previous work in our laboratory has shown that the transcription of the Tgase gene is directly controlled by retinoic acid and retinoic acid receptors. To identify the region of the Tgase gene responsible for regulating its expression, fragments of the Tgase promoter and 5$\sp\prime$-flanking region were cloned into the chloramphenicol actetyl transferase (CAT) reporter constructs. Transient transfection experiments with these constructs demonstrated that the upstream region of Tgase is a functional promoter which contains a retinoid response element within a 1573 nucleotide region spanning nucleotides $-$252 to $-$1825. ^

3D MPRAGE improves classification of cortical lesions in multiple sclerosis.

BACKGROUND: Gray matter lesions are known to be common in multiple sclerosis (MS) and are suspected to play an important role in disease progression and clinical disability. A combination of magnetic resonance imaging (MRI) techniques, double-inversion recovery (DIR), and phase-sensitive inversion recovery (PSIR), has been used for detection and classification of cortical lesions. This study shows that high-resolution three-dimensional (3D) magnetization-prepared rapid acquisition with gradient echo (MPRAGE) improves the classification of cortical lesions by allowing more accurate anatomic localization of lesion morphology. METHODS: 11 patients with MS with previously identified cortical lesions were scanned using DIR, PSIR, and 3D MPRAGE. Lesions were identified on DIR and PSIR and classified as purely intracortical or mixed. MPRAGE images were then examined, and lesions were re-classified based on the new information. RESULTS: The high signal-to-noise ratio, fine anatomic detail, and clear gray-white matter tissue contrast seen in the MPRAGE images provided superior delineation of lesion borders and surrounding gray-white matter junction, improving classification accuracy. 119 lesions were identified as either intracortical or mixed on DIR/PSIR. In 89 cases, MPRAGE confirmed the classification by DIR/PSIR. In 30 cases, MPRAGE overturned the original classification. CONCLUSION: Improved classification of cortical lesions was realized by inclusion of high-spatial resolution 3D MPRAGE. This sequence provides unique detail on lesion morphology that is necessary for accurate classification.

Automatic quantitation of multiple sclerosis lesions on MR images

A two-pronged approach for the automatic quantitation of multiple sclerosis (MS) lesions on magnetic resonance (MR) images has been developed. This method includes the design and use of a pulse sequence for improved lesion-to-tissue contrast (LTC) and seeks to identify and minimize the sources of false lesion classifications in segmented images. The new pulse sequence, referred to as AFFIRMATIVE (Attenuation of Fluid by Fast Inversion Recovery with MAgnetization Transfer Imaging with Variable Echoes), improves the LTC, relative to spin-echo images, by combining Fluid-Attenuated Inversion Recovery (FLAIR) and Magnetization Transfer Contrast (MTC). In addition to acquiring fast FLAIR/MTC images, the AFFIRMATIVE sequence simultaneously acquires fast spin-echo (FSE) images for spatial registration of images, which is necessary for accurate lesion quantitation. Flow has been found to be a primary source of false lesion classifications. Therefore, an imaging protocol and reconstruction methods are developed to generate "flow images" which depict both coherent (vascular) and incoherent (CSF) flow. An automatic technique is designed for the removal of extra-meningeal tissues, since these are known to be sources of false lesion classifications. A retrospective, three-dimensional (3D) registration algorithm is implemented to correct for patient movement which may have occurred between AFFIRMATIVE and flow imaging scans. Following application of these pre-processing steps, images are segmented into white matter, gray matter, cerebrospinal fluid, and MS lesions based on AFFIRMATIVE and flow images using an automatic algorithm. All algorithms are seamlessly integrated into a single MR image analysis software package. Lesion quantitation has been performed on images from 15 patient volunteers. The total processing time is less than two hours per patient on a SPARCstation 20. The automated nature of this approach should provide an objective means of monitoring the progression, stabilization, and/or regression of MS lesions in large-scale, multi-center clinical trials. ^

PHENOTYPIC ASSOCIATION OF GENE AMPLIFICATION WITH MULTIPLE DRUG RESISTANCE IN VINCRISTINE RESISTANT CHINESE HAMSTER OVARY CELLS

Resistance of tumors to pharmacologic agents poses a significant problem in the treatment of human malignancies. This study overviews the scope of clinical resistance and focuses upon current research attempts toward investigation of the phenomenon of multidrug resistance (MDR).^ The objective of this investigation was to determine whether gene amplification had a role in the development of the MDR phenotype in Chinese hamster ovary cells (CHO) primarily selected for resistance to vincristine (VCR). A DNA fragment, previously shown to be amplified in two independently derived Chinese hamster cell lines exhibiting the MDR phenotype, was also amplified in VCR hamster lines. Sequences flanking this fragment were shown to contain coding information for a 4.3 kb transcript overproduced in VCR cells. These sequences were not enriched in double minute DNA preparations isolated from VCR cells. There was an approximately forty-fold increase in both the level of gene amplification and transcript overproduction in the VCR cell lines, independent of the level of primary resistance. This DNA amplification and overproduction of the 4.3 kb transcript was also demonstrated in CHO cells independently selected for resistance to Adriamycin and vinblastine.^ All the DNA sequences of two hamster cDNA clones containing 785 and 932 base pair inserts showed direct homology to the published mouse mdr sequences (about 90%). This sequence conservation held for only portions of the gene when the human mdr1 sequences were compared with those from either the mouse or hamster.^ Somatic cell hybrids, constructed between VCR CHO cells and sensitive murine cells, were used to determine whether there was a functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of MDR-associated amplified sequences, overexpression of the mRNA encoded by these sequences, overexpression of the mRNA encoded by these sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the VCR CHO cells which is responsible for MDR in these cells. ^

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

Multiple gradient echo propeller (MGREP) MRI: Technical development and potential applications

The PROPELLER (Periodically Rotated Overlapping Parallel Lines with Enhanced Reconstruction) magnetic resonance imaging (MRI) technique has inherent advantages over other fast imaging methods, including robust motion correction, reduced image distortion, and resistance to off-resonance effects. These features make PROPELLER highly desirable for T2*-sensitive imaging, high-resolution diffusion imaging, and many other applications. However, PROPELLER has been predominantly implemented as a fast spin-echo (FSE) technique, which is insensitive to T2* contrast, and requires time-inefficient signal averaging to achieve adequate signal-to-noise ratio (SNR) for many applications. These issues presently constrain the potential clinical utility of FSE-based PROPELLER. ^ In this research, our aim was to extend and enhance the potential applications of PROPELLER MRI by developing a novel multiple gradient echo PROPELLER (MGREP) technique that can overcome the aforementioned limitations. The MGREP pulse sequence was designed to acquire multiple gradient-echo images simultaneously, without any increase in total scan time or RF energy deposition relative to FSE-based PROPELLER. A new parameter was also introduced for direct user-control over gradient echo spacing, to allow variable sensitivity to T2* contrast. In parallel to pulse sequence development, an improved algorithm for motion correction was also developed and evaluated against the established method through extensive simulations. The potential advantages of MGREP over FSE-based PROPELLER were illustrated via three specific applications: (1) quantitative T2* measurement, (2) time-efficient signal averaging, and (3) high-resolution diffusion imaging. Relative to the FSE-PROPELLER method, the MGREP sequence was found to yield quantitative T2* values, increase SNR by ∼40% without any increase in acquisition time or RF energy deposition, and noticeably improve image quality in high-resolution diffusion maps. In addition, the new motion algorithm was found to improve the performance considerably in motion-artifact reduction. ^ Overall, this work demonstrated a number of enhancements and extensions to existing PROPELLER techniques. The new technical capabilities of PROPELLER imaging, developed in this thesis research, are expected to serve as the foundation for further expanding the scope of PROPELLER applications. ^

Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^