Time and biosequences

In this paper we discuss and demonstrate the importance of several factors relative to the relationship between time and evolution of biosequences. In both quantitative and qualitative measurements of the genetic distances, the compositional constraints of the nucleotide sequences play a very important role. We demonstrate that when homologous sequences significantly differ in base composition we get erratic branching order and/or wrong evaluation of the evolutionary rates. We must consider that every gene may have a different evolutionary dynamic along its sequence, generally linked to its functional constraints; this too can seriously affect its clocklike behavior. We report some cases showing how these factors can affect the quantitative measurements of the genetic distances of biosequences. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Enhanced identification of peptides lacking basic residues by LC-ESI-MS/MS analysis of singly charged peptides

Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as "base-less") are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/M Sanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MS schemes. In the present experimental outcome, additional LC-MS/MS analysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MS based identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.     

Excision Repair of 2,5-Diaziridinyl-1,4-Benzoquinone (DZQ)-DNA Adduct by Bacterial and Mammalian 3-Methyladenine-DNA Glycosylases

The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formami-dopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.     

Colon cancer-associated DNA polymerase β variant induces genomic instability and cellular transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.     

Effect of temperature and cold stratification on seed germination of the Mediterranean wild aromatic Clinopodium sandalioticum (Lamiaceae)

A germination study was carried out on seeds of Clinopodium sandalioticum (Bacch. & Brullo) Bacch. & Brullo ex Peruzzi & Conti (Lamiaceae), a wild aromatic plant endemic to Sardinia. Seeds were incubated at a range of constant (5–25°C) and an alternating temperatures regime (25/10°C), with 12 hours of irradiance per day. The results achieved at 10°C were also compared with those obtained after a period of cold stratification at 5°C for three months. Final seed germination ranged from ca. 28% (5°C) to ca. 72% (25/10°C). A base temperature for germination (T_b) of ca. 5°C and a thermal constant for 50% germination (S) of 89.3°Cd were identified and an optimal temperature for germination (T_o) was estimated to be comprised between 20 and 25°C. Cold stratification negatively affected seed viability and germination at 10°C. Although a typical “Mediterranean germination syndrome”, could not be detected for C. sandalioticum seeds, these results were coherent with those previously reported for other Mediterranean Lamiaceae species.     

Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites

Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, methyl binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G·T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.     

不同基肥对退化坡地及牧草产量和质量的影响

探讨了不同基肥组合处理对退化坡地的改造利用和对牧草生产的影响．在相同的肥水管理条件下,以“鸡粪＋牛粪＋石灰”的基肥组合（Ａ）处理退化坡地土壤,可使皇草、杂交狼尾草、矮象草和墨西哥玉米的年产量比“猪粪＋牛粪＋石灰”的组合（Ｂ）处理分别高出９．７％、１２．８％、４３．０％和９．９％,前者（Ａ）生产出的牧草的粗蛋白含量略高于后者（Ｂ）,粗纤维含量略低于后者,而粗灰分含量显著高于后者．     

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.