p63 mutations have been identified in several developmental abnormalities, including split-hand/foot malformation (SHFM). In this study, we demonstrate that the C-terminal domain of p63alpha ...associates with the E2 ubiquitin conjugating enzyme, Ubc9. A p63alpha mutation, Q634X, which naturally occurs in SHFM modulated the interaction of p63alpha with Ubc9 in yeast genetic assay. Furthermore, Ubc9 catalyzed the conjugation of p63alpha with small ubiquitin modifier-1 (SUMO-1), which covalently modified p63alpha in vitro and in vivo at two positions (K549E and K637E), each situated in a SUMO-1 modification consensus site (phiKXD/E). In addition, p63alpha mutations (K549E and K637E) abolished sumoylation of p63alpha, dramatically activated transactivation properties of TAp63alpha, and inhibited the dominant-negative effect of DeltaNp63alpha. These p63alpha mutations also affected the transcriptional regulation of gene targets involved in bone and tooth development (e.g., RUNX2 and MINT) and therefore might contribute to the molecular mechanisms underlying the SHFM phenotype.
p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using ...oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63
+/+
mouse, it is undetectable in these tissues in the p63
−/−
mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.
A new E.coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methyla-tion-dependent restriction systems (McrA−;, McrBC−, Mrr−;). This strain has been used to ...clone restriction endonuclease genes directly into E.coli. When E.coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes TaqI (5′TCGA3′) and Tth111I (5′GACNNNGTC3′) have been successfully cloned in E.coli. The new strain will be useful to clone other genes involved in DNA metabolism.
A gene coding for a thermostable nuclease was cloned from the thermophilic microorganism,
Thermusfiliformis (Tf), using an indicator strain containing a
dinD∷lacZ fusion. The gene, designated
nuc17, ...has been mapped within a 2300-bp fragment. The 55-kDa
Tf nuclease was purified to over 95% homogeneity. Single-stranded (ss) DNA is the preferred substrate for the
Tfnuclease, although double-stranded (ds) DNA can also be digested. Nuclease activity increases with increasing temperature up to 80°C and requires the metal ions Ca
+− or Mg
++for catalysis.
Tf nuclease is primarily an endonuclease that leaves 5′ phosphates in the digested products. The ssDNA extensions remaining after exonuclease III digestion of dsDNA can be removed by the
Tf nuclease, making it a useful reagent to generate unidirectional deletions.
Two heat-sensitive
R.BamHI mutants, T1571 and P173L, and one cold-sensitive
R.BamHI mutant, T1141, were isolated after chemical mutagenesis of the
bamhIR gene that codes for the restriction ...endonuclease
BamHI (R.BamHI). The thermosensitivity of T1141, T1571 and P173L is revealed by the 10
2−10
3 lower plating efficiency at the non-permissive temperature of strains bearing these alleles. The conditional-lethal phenotype can be rescued by introduction of the cognate
bamhIM gene into the same cell. The mutant enzymes induce the SOS response in vivo and display reduced phage restriction activity. The P173L protein, when expressed at 30°C and purified, shows reduced thermostability at 65°C. T1571 and P173L mutants yield different intermediates during partial trypsin digestion. The conditional-lethal
BamHI mutants could be used to deliver in vivo DNA cleavage and for further isolation of relaxed-specificity mutants.
A 1440-bp plasmid named pAP12875 was isolated from
Acetobacter pasteurianus and its nucleotide sequence determined. An open reading frame was found capable of coding for a protein that has similarity ...with the replication protein of pVT736-1 from
Actinobacillus actinomycetemcomitans and the 32-kDa protein of phage Pf3 from
Pseudomonas aeruginosa
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