Mary L. Durbin

Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

Cellulases in Phaseolus vulgaris

Publisher Summary Research on cellulase in the bean plant, Phaseolus vulgaris L. cv. Red Kidney, has led to the discovery of a family of cellulases with differing forms and functions. The development of methods for differentiating cellulase activities made it possible to begin sorting out the functions of the various cellulases. With the purification of the 9.5 cellulase and subsequent production of antibodies to it, the amount of 9.5 cellulase activity vs acidic pl cellulases could be quantitated. It was found that 9.5 cellulase is synthesized de novo in the abscission zone of bean leaves in response to ethylene. Synthesis of 9.5 cellulase is confined to a very narrow band of cells in the abscission zone. The acidic forms of cellulase occur throughout the plant, particularly in young rapidly expanding tissue. These forms appear to be auxin regulated and involved in growth and differentiation. The affinity of 9.5 cellulase has been exploited for its substrate and purified the enzyme on a cellulose column. Hydrolysis of the cellulose does occur to some extent, as reducing sugars are found in the peak fractions containing the purified enzyme. As the acidic cellulases have only been partially purified, their substrate specificity has not been firmly established.

Genes that determine flower color: the role of regulatory changes in the evolution of phenotypic adaptations

A central goal of evolutionary genetics is to trace the causal pathway between mutations at particular genes and adaptation at the phenotypic level. The proximate objective is to identify adaptations through the analysis of molecular sequence data from specific candidate genes or their regulatory elements. In this paper, we consider the molecular evolution of floral color in the morning glory genus (Ipomoea) as a model for relating molecular and phenotypic evolution. To begin, flower color variation usually conforms to simple Mendelian transmission, thus facilitating genetic and molecular analyses. Population genetic studies of flower color polymorphisms in the common morning glory (Ipomoea purpurea) have shown that some morphs are subject to complex patterns of selection. Striking differences in floral color and morphology are also associated with speciation in the genus Ipomoea. The molecular bases for these adaptive shifts can be dissected because the biosynthetic pathways that determine floral pigmentation are well understood and many of the genes of flavonoid biosynthesis have been isolated and extensively studied. We present a comparative analysis of the level of gene expression in Ipomoea for several key genes in flavonoid biosynthesis. Specifically we ask: how frequently are adaptive shifts in flower color phenotypes associated with changes in regulation of gene expression versus mutations in structural genes? The results of this study show that most species differences in this crucial phenotype are associated with changes in the regulation of gene expression.

Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4α (HNF4α) gene

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4α1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4α gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4α protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4α P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4α P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6α. p53 bound HNF6α in vivo and in vitro and prevented HNF6α from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6α in vivo. However, since the R249S p53 mutant also bound HNF6α, binding HNF6α is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4α expression in response to cellular stress are discussed.

Nucleotide polymorphism in the chalcone synthase‐A locus and evolution of the chalcone synthase multigene family of common morning glory Ipomoea purpurea

Chalcone synthase (CHS) is a small multigene family with at least four members (CHS‐A, B, C and PS) in common morning glory Ipomoea purpurea ROTH. The chalcone synthase enzyme performs the initial condensation reaction that results in the 15‐carbon three‐ring structure that is the backbone of flavonoid biosynthesis. The biochemical pathway that commences with CHS is important in plant disease defence, pigment biosynthesis and UV protection. Accordingly, it is of substantial interest to characterize levels and patterns of molecular diversity for genes that encode this important enzyme. We report the sequence of 19 CHS‐A alleles from Mexican and American populations of common morning glory. American populations of this annual self‐compatible vine are believed to have been introduced from Mexico, where the species is native. Individual plants were sampled from populations of common morning glory throughout Mexico and the south‐eastern USA. Four American alleles were sequenced and these, together with one allele from Mexico City, were identical in primary nucleotide sequence. These data suggest a restricted origin for the American population, probably as a consequence of selection for domestication by pre‐Columbian peoples. Additionally the Mitontic (Chiapas, Mexico) population is significantly more homogeneous than expected by chance indicating that this population may also have experienced a recent population bottleneck. Estimates of nucleotide diversity from the Mexican CHS‐A alleles were high. We present evidence that these estimates may, in part, result from low to moderate levels of interlocus recombination/gene conversion. We also present evidence that the ancient duplication of the CHS gene family, preceding the origin of the genus Ipomoea, was associated with heterogeneity in the rate of substitution between the resulting gene family members. The group of gene family members whose sequences possess a signature amino acid of the closely related Stilbene synthase exhibit a significantly faster proportional rate of nonsynonymous substitution.

The immunocytochemical localization of 9.5 cellulase in abscission zones of bean (Phaseolus vulgaris cv. red kidney)

SummaryAntibodies raised against 9.5 cellulase (β 1,4 glucan-4-glucanohydrolase) were used to investigate the distribution of the enzyme in abscising bean petioles (Phaseolus vulgaris L. cv. Red Kidney).Results showed the enzyme to be localized in the cortical separation layer, which is 2 or 3 cells wide; this distribution is consistent with the proposed role of cellulase in abscission. Unfortunately, the enzymes distribution in the stele was masked by non-specific staining. Direct assay of small fragments dissected from the abscission zone confirmed these findings and also showed very high activity levels in the stele. The vascular cellulase spread acropetally from the separation zone some 4 mm into the pulvinus. Experiments isolating the distal pulvinus prior to induction of abscission revealed that this activity was not the result of diffusion of cellulase from the separation layer.

Purification of a cellulase from kidney bean abscission zones

Abstract The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single 14C-labeled protein from an abscission zone extract heavily labeled with 14C amino acids.

Pulmonary adenocarcinoma–targeted gene therapy by a cancer- and tissue-specific promoter system

Gene therapy is one of the approaches used to treat lung cancer. The benefit of cancer gene therapy is that different types of tumors can be selectively targeted by tumor-specific expression of therapeutic genes that include an apoptosis gene to destroy the tumor. Previously, we described a promoter (TTS promoter) that we designed that is specifically targeted to lung cancer cells but not to other types of cancer or normal cells including stem cells. In this pursuit, we further characterize the specificity of the TTS promoter in four types of lung cancer cells (squamous cell lung carcinoma, pulmonary adenocarcinoma, small-cell lung carcinoma, large-cell lung carcinoma). The TTS promoter is highly active only in pulmonary adenocarcinoma cells but not in the other three types of lung cancer cells. The specificity seems to be derived from transcription factor thyroid transcription factor 1–associating cofactors that affect human surfactant protein A1 promoter activity in pulmonary adenocarcinoma. We inserted the proapoptotic gene Bcl-2–associated X protein (Bax) into the TTS promoter (TTS/Bax). The TTS/Bax selectively causes BAX expression and cell death in pulmonary adenocarcinoma but not in other cells. Cell death caused by the BAX expression was also observed in pulmonary adenocarcinoma that is resistant to the anticancer drug gefitinib (epidermal growth factor receptor tyrosine kinase inhibitor). BAX expression and cell death can be suppressed by dexamethasone (a glucocorticoid) treatment through negative glucocorticoid elements in the TTS promoter. Here we report a drug-controllable TTS/Bax system targeting pulmonary adenocarcinoma. [Mol Cancer Ther 2007;6(1):244–52]

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

Malignant Pleural Mesothelioma–Targeted CREBBP/EP300 Inhibitory Protein 1 Promoter System for Gene Therapy and Virotherapy

Gene therapy and virotherapy are one of the approaches used to treat malignant pleural mesothelioma. To improve the efficiency of targeting malignant mesothelioma cells, we designed a novel system using the promoter of the CREBBP/EP300 inhibitory protein 1 (CRI1), a gene specifically expressed in malignant pleural mesothelioma. Four tandem repeats of the CRI1 promoter (CRI1(-138 4x)) caused significantly high promoter activity in malignant pleural mesothelioma cells but little promoter activity in normal mesothelial cells and normal fibroblasts. The recombinant adenoviral vector expressing proapoptotic BH3-interacting death agonist or early region 1A driven by the CRI1(-138 4x) promoter induced cell death in malignant mesothelioma cells but not in normal cells. Moreover, these viruses showed antitumor effects in a mesothelioma xenograft mouse model. Here, we describe a novel strategy to target malignant mesothelioma using the CRI1(-138 4x) promoter system.