Mutations affecting beta-tubulin folding and degradation.

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Mutations in myosin light chain kinase cause familial aortic dissections.

Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections.

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A- and B-subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT-PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.

Contribution of Ectodomain Mutations in Epidermal Growth Factor Receptor to Signaling in Glioblastoma Multiforme

CONTRIBUTION OF ECTODOMAIN MUTATIONS IN EPIDERMAL GROWTH FACTOR RECEPTOR TO SIGNALING IN GLIOBLASTOMA MULTIFORME Publication No._________ Marta Rojas, M.S. Supervisory Professor: Oliver Bögler, Ph.D. The Cancer Genome Atlas (TCGA) has conducted a comprehensive analysis of a large tumor cohort and has cataloged genetic alterations involving primary sequence variations and copy number aberrations of genes involved in key signaling pathways in glioblastoma (GBM). This dataset revealed missense ectodomain point mutations in epidermal growth factor receptor (EGFR), but the biological and clinical significance of these mutations is not well defined in the context of gliomas. In our study, we focused on understanding and defining the molecular mechanisms underlying the functions of EGFR ectodomain mutants. Using proteomic approaches to broadly analyze cell signaling, including antibody array and mass spectrometry-based methods, we found a differential spectrum of tyrosine phosphorylation across the EGFR ectodomain mutations that enabled us to stratify them into three main groups that correlate with either wild type EGFR (EGFR) or the long-studied mutant, EGFRvIII. Interestingly, one mutant shared characteristics of both groups suggesting a continuum of behaviors along which different mutants fall. Surprisingly, no substantial differences were seen in activation of classical downstream signaling pathways such as Akt and S6 pathways between these classes of mutants. Importantly, we demonstrated that ectodomain mutations lead to differential tumor growth capabilities in both in vitro (anchorage independent colony formation) and in vivo conditions (xenografts). Our data from the biological characterization allowed us to categorize the mutants into three main groups: the first group typified by EGFRvIII are mutations with a more aggressive phenotype including R108K and A289T; a second group characterized by a less aggressive phenotype exemplified by EGFR and the T263P mutation; and a third group which shared characteristics from both groups and is exemplified by the mutation A289D. In addition, we treated cells overexpressing the mutants with various agents employed in the clinic including temozolomide, cisplatin and tarceva. We found that cells overexpressing the mutants in general displayed resistance to the treatments. Our findings yield insights that help with the molecular characterization of these mutants. In addition, our results from the drug studies might be valuable in explaining differential responses to specific treatments in GBM patients.

ROLES FOR BRAF KINASE ACTIVATING MUTATIONS IN MELANOMA: MICROENVIRONMENTAL IMMUNOSUPPRESSION

The BRAF oncogene demonstrates a characteristic mutation (V600E) in a significant fraction of cutaneous melanomas, leading to constitutive activation of the MAP kinase pathway. This genetic lesion endows tumor cells with proliferative and survival advantages, and metastatic melanoma patients treated with the BRAF(V600E)-specific inhibitor, Vemurafenib, have shown dramatic clinical responses. Here, I show that BRAF(V600E) induces transcription of the IL-1α and IL-1β genes in both melanocytes and melanoma cell lines and that this upregulation is specifically abrogated by targeted BRAF(V600E) inhibitors. Furthermore, treatment of melanoma tumor-associated fibroblasts (TAFs) with IL-1α/β significantly enhanced the ability of TAFs to suppress the proliferation and function of melanoma antigen-specific cytotoxic T cells. IL-1α/β treatment of TAFs upregulated multiple immunosuppressive factors, including COX-2 and the PD-1 ligands PD-L1 and PD-L2. Specific BRAF(V600E) inhibitors largely abrogated the ability of melanoma cells to confer T cell-suppressive properties on TAFs. These results support a model in which BRAF(V600E) promotes immune suppression in the melanoma tumor environment through an IL-1-mediated mechanism involving resident stromal fibroblasts. Based on these findings, combination therapies involving targeted BRAF inhibition and T cell-based immunotherapies are warranted.

Isolation and analysis of regulatory and structural mutations in the {\it recA\/} gene of {\it Escherichia coli}

The recA gene is essential for homologous recombination and for inducible DNA repair in Escherichia coli. The level of recA expression is important for these functions. The growth defect of a lambda phage carrying a recA-lacZ fusion was used to select mutations that reduced recA expression. Nine of these mutations were single base changes in the recA promoter; each reduced both induced and basal (repressed) levels of expression, indicating that only one promoter is used under both circumstances. Deletion analysis of the promoter region and S1 mapping of transcripts confirmed that there is only one promoter responsible for both basal and induced expression. Some of the mutants, however, displayed a ratio of induced to repressed expression that was much lower than wild-type. For one of these mutants (recA1270) LexA binding studies showed that this was not due to a change in the affinity of LexA repressor for the operator site. The extent of binding of RNA polymerase to this mutant promoter, however, was much reduced, and the complexes formed were qualitatively different. Further binding experiments provided some evidence that LexA does not block RNA polymerase binding to the recA promoter, but inhibits a later step in initiation. Behavior of the mutants with altered induction ratios could be explained if LexA binding to the operator actually increases RNA polymerase binding to the promoter in a closed complex compensating for defects in polymerase binding caused by the mutations.^ In a study of mutations in the recA structural gene, site-directed mutagenesis was used to replace cysteine codons at positions 90, 116, and 129 with a number of different codons. In vivo analysis of the replacements showed that none of the cysteines is absolutely essential and that they do not have a direct role as catalysts in ATP hydrolysis. Some amino acid substitutions abolished all RecA functions, while a few resulted in partial or altered function. Amino acids at positions 90 and 129 tended to affect all functions equally, while the amino acid at position 116 appeared to have a particular effect on the protease activity of the protein. ^

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

Role of germ line p53 mutations in aneuploidy and immortalization of dermal fibroblasts from Li-Fraumeni cancer patients

Pedigree analysis of certain families with a high incidence of tumors suggests a genetic predisposition to cancer. Li and Fraumeni described a familial cancer syndrome that is characterized by multiple primary tumors, early age of onset, and marked variation in tumor type. Williams and Strong (1) demonstrated that at least 7% of childhood soft tissue sarcoma patients had family histories that is readily explained by a highly penetrant autosomal dominant gene. To characterize the mechanism for genetic predisposition to many tumor types in these families, we have studied genetic alterations in fibroblasts, a target tissue from patients with the Li-Fraumeni Syndrome (LFS).^ We have observed spontaneous changes in initially normal dermal fibroblasts from LFS patients as they are cultured in vitro. The cells acquire an altered morphology, chromosomal anomalies, and anchorage-independent growth. This aberrant behavior of fibroblasts from LFS patients had never been observed in fibroblasts from normal donors. In addition to these phenotypic alterations, patient fibroblasts spontaneously immortalize by 50 population doublings (pd) in culture; unlike controls that remain normal and senesce by 30-35 (2). At 50 pd, immortal fibroblasts from two patients were found to be susceptible to tumorigenic transformation by an activated T24 H-ras oncogene (3). Approximately 80% of the oncogene expressing transfectants were capable of forming tumors in nude mice within 2-3 weeks. p53 has been previously associated with immortalization of cells in culture and cooperation with ras in transfection assays. Therefore, patients' fibroblast and lymphocyte derived DNA was tested for point mutations in p53. It was shown that LFS patients inherited certain point mutations in one of the two p53 alleles (4). Further studies on the above LFS immortal fibroblasts have demonstrated loss of the remaining p53 allele concomitant with escape from senescence. While the loss of the second allele correlates with immortalization it is not sufficient to transformation by an activated H-ras or N-ras oncogene. These immortal fibroblasts are resistant to tumorigenic transformation by v-abl, v-src, c-neu or v-mos oncogene; implying that additional steps are required in the tumorigenic progression of LFS patients' fibroblasts.^ References. (1) Williams et al., J. Natl. Cancer Inst. 79:1213, 1987. (2) Bischoff et al., Cancer Res. 50:7979, 1990. (3) Bischoff et al., Oncogene 6:183, 1991. (4) Malkin et al., Science 250:1233, 1990. ^

Identification of cadmium-induced mutations in a mammalian cell line

Epidemiological studies have shown cadmium to induce cancer in humans, while experimental studies have proven this metal to be a potent tumor inducer in animals. However, cadmium appears nonmutagenic in most prokaryotic and eukaryotic mutagenesis assays. In this study, we present the identification of mutations in normal rat kidney cells infected with the mutant MuSVts110 retrovirus (6m2 cells) as a result of treatment with cadmium chloride. The detection of these mutations was facilitated by the use of a novel mutagenesis assay established in this laboratory. The 6m2 reversion assay is a positive selection system based on the conditional expression of the MuSVts110 v-mos gene. In MuSVts110 the gag and mos genes are fused out of frame, thus the translation of the v-mos sequence requires a frameshift in the genomic RNA. In 6m2 cells this frameshift is accomplished by the temperature-dependent splicing of the primary MuSVts110 transcript. Splicing of MuSVts110, which is mediated by cis-acting sequences, occurs when 6m2 cells are grown at 33$\sp\circ$C and below, but not at 39$\sp\circ$C. Therefore, 6m2 cells appear transformed at low growth temperatures, but take on a morphologically normal appearance when grown at high temperatures. The treatment of 6m2 cells with cadmium chloride resulted in the outgrowth of a number of cells that reverted to the transformed state at high growth temperatures. Analysis of the viral proteins expressed in these cadmium-induced 6m2 revertants suggested that they contained mutations in their MuSVts110 DNA. Sequencing of the viral DNA from three revertants that constitutively expressed the P85$\sp{gag{-}mos}$ transforming protein revealed five different mutations. The Cd-B2 revertant contained three of those mutations: an A-to-G transition 48 bases downstream of the MuSVts110 3$\sp\prime$ splice site, plus a G-to-T and an A-to-T transversion 84 and 100 bases downstream of the 5$\sp\prime$ splice site, respectively. The Cd-15-5 revertant also contained a point mutation, a T-to-C transition 46 bases downstream of the 5$\sp\prime$ splice site, while Cd-10-5 contained a three base deletion of MuSVts110 11 bases upstream of the 3$\sp\prime$ splice site. A fourth revertant, Cd-10, expressed a P100$\sp{gag{-}mos}$ transforming protein, and was found to have a two base deletion. This deletion accomplished the frameshift necessary for v-mos expression, but did not alter MuSVts110 RNA splicing and the expression of p85$\sp{gag{-}mos}.$ Lastly, sequencing of the MuSVts110 DNA from three spontaneous revertants revealed the same G to T transversion in each one. This was the same mutation that was found in the Cd-B2 revertant. These findings provide the first example of mutations resulting from exposure to cadmium and suggest, by the difference in each mutation, the complexity of the mechanism utilized by cadmium to induce DNA damage. ^

Independent polled mutations leading to complex gene expression differences in cattle

The molecular regulation of horn growth in ruminants is still poorly understood. To investigate this process, we collected 1019 hornless (polled) animals from different cattle breeds. High-density SNP genotyping confirmed the presence of two different polled associated haplotypes in Simmental and Holstein cattle co-localized on BTA 1. We refined the critical region of the Simmental polled mutation to 212 kb and identified an overlapping region of 932 kb containing the Holstein polled mutation. Subsequently, whole genome sequencing of polled Simmental and Holstein cows was used to determine polled associated genomic variants. By genotyping larger cohorts of animals with known horn status we found a single perfectly associated insertion/deletion variant in Simmental and other beef cattle confirming the recently published possible Celtic polled mutation. We identified a total of 182 sequence variants as candidate mutations for polledness in Holstein cattle, including an 80 kb genomic duplication and three SNPs reported before. For the first time we showed that hornless cattle with scurs are obligate heterozygous for one of the polled mutations. This is in contrast to published complex inheritance models for the bovine scurs phenotype. Studying differential expression of the annotated genes and loci within the mapped region on BTA 1 revealed a locus (LOC100848215), known in cow and buffalo only, which is higher expressed in fetal tissue of wildtype horn buds compared to tissue of polled fetuses. This implicates that the presence of this long noncoding RNA is a prerequisite for horn bud formation. In addition, both transcripts associated with polledness in goat and sheep (FOXL2 and RXFP2), show an overexpression in horn buds confirming their importance during horn development in cattle.