Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

pORE: a modular binary vector series suited for both monocot and dicot plant transformation

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an ‘open’ set for general plant transformation, a ‘reporter’ set for promoter analysis and an ‘expression’ set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P _HPL, P _ENTCUP2 and P _TAPADH), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).     

Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication

Summary A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the — 35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

The tomato early fruit specific gene <Emphasis Type="Italic">Lefsm1</Emphasis> defines a novel class of plant-specific SANT/MYB domain proteins

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.