We have yet to develop a fundamental understanding of the molecular complexities of human spermatozoa. This encompasses the unique packaging and structure of the sperm genome along with their paternally derived RNAs in preparation for their delivery to the egg. The diversity of these transcripts is vast, including several anti-sense molecules resembling known regulatory micro-RNAs. The field is still grasping with its delivery to the oocyte at fertilization and possible significance. It remains tempting to analogize them to maternally-derived transcripts active in early embryo patterning. Irrespective of their role in the embryo, their use as a means to assess male factor infertility is promising.
DNA ; genetics ; metabolism ; Humans ; Male ; RNA, Messenger ; genetics ; Spermatozoa ; metabolism

DNA Damage ; DNA Glycosylases ; genetics ; metabolism ; DNA Repair Enzymes ; genetics ; metabolism ; Humans ; Phosphoric Monoester Hydrolases ; genetics ; metabolism

OBJECTIVEExpress a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.

METHODSWe express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.

RESULTSThe plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.

CONCLUSIONSkod-ssb may in future be used to enhance DNA and cDNA amplification.

Archaeal Proteins ; genetics ; isolation & purification ; metabolism ; Chromatography, Affinity ; DNA, Bacterial ; genetics ; metabolism ; DNA, Complementary ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Thermococcus ; genetics ; metabolism

Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these iron-requiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.
Animals ; Cell Cycle Checkpoints ; DNA ; metabolism ; DNA Repair ; DNA Replication ; Hemeproteins ; genetics ; metabolism ; Iron ; chemistry ; metabolism ; Iron-Sulfur Proteins ; genetics ; metabolism ; Ribonucleotide Reductases ; genetics ; metabolism ; Yeasts ; metabolism

OBJECTIVES: To explore the correlation between cytosine-phosphoric-guanylic (CpG) site of Septin 9 gene and colorectal cancer, and to develop a real-time PCR detection system in plasma in patients with colorectal cancer. METHODS: The methylation of training samples was detected by high-throughput sequencing technology, and the sites highly consistent with the clinical information of colorectal cancer were identified. Then the detection system of real-time PCR was designed to analyze the consistency of plasma and tissue based on methylationa sensitive enzyme digestion. Finally, 100 clinical trials were conducted to evaluate the performance of the detection system with the methylation sensitive enzyme digestion-real-time PCR. RESULTS: The highly consistent sites, which were selected by high-throughput sequencing from 71 training set samples, was the 38th CpG. Based on the detection region, the screened methylation sensitive enzymes were CONCLUSIONS The 38th CpG site of Septin 9 detected by the detection system of methylation sensitive enzyme digestion-real-time PCR can highly predict the occurrence of colorectal cancer with great clinical application value.
Colorectal Neoplasms/genetics* ; CpG Islands/genetics* ; DNA ; DNA Methylation ; Humans ; Plasma/metabolism* ; Septins/metabolism*

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Restriction Enzymes ; metabolism ; DNA, Neoplasm ; genetics ; Glutathione S-Transferase pi ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Polymerase Chain Reaction ; methods

Erasure, establishment and maintenance of genetic imprinting are indispensable for normal embryonic development. All these processes depend on accurate expression and intimate cooperation of kinds of DNA methyltransferases. Many genetic syndromes and embryo developmental anomalies are caused by abnormality of genetic imprinting. Genetic imprinting is important for the nucleus totipotential of primordial germ cell, maturation of gamete,growth and development of embryo, structure and function of placenta as well as postnatal growth and development of individuals.
Animals ; DNA Methylation ; DNA Modification Methylases ; genetics ; metabolism ; Embryonic Development ; genetics ; Genomic Imprinting ; genetics ; Humans ; Mutation

The disorders of DNA and histone methylation have a close relationship with the development and progression of tumors. Epigenetic regulation is critical in maintaining the stability and integrity of the expression profiles of different cell types by modifying DNA methylation and histone methylation. However, the abnormal changes of methylation often result in the development and progression of tumors. This review summarized the theory of tumor genomic and histone methylation, detection methods of methylation and their applications, and the clinical application of methylation as biological markers and drug targets.
DNA Methylation ; Histones ; metabolism ; Humans ; Methylation ; Neoplasms ; genetics ; metabolism

Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.
Sirtuins/genetics/*metabolism ; RNA, Small Interfering/genetics ; Humans ; DNA-Binding Proteins/*metabolism ; DNA Repair/*genetics ; DNA/*genetics ; Cell Line ; Antigens, Nuclear/*metabolism ; Acetylation

To explore the role of the Chk2 protein expression and DNA double strand breaks (DSBs) repair in low dose hyper-radiosensitivity (HRS)/increased radioresistance (IRR) of non-small cell lung cancer, A549 cells were subjected to irradiation at the dosage ranging from 0.05-2 Gy. Clonogenic survival was measured by using fluorescence-activated cell sorting (FACS) plating technique. Percentage of cells in M-phase after low doses of X-irradiation was evaluated by phospho-histone H3-FITC/PI and Western blotting was used to detect protein expression of Chk2 and phospo-Chk2. DNA DSBs repair efficiency was also measured by induction and persistence of γ-H2AX. The results showed that the killing ability of irradiation with A549 cells increased at low conditioning dose below 0.3 Gy. Within the dose of 0.3 to 0.5 Gy, A549 cells showed a certain extent of radiation resistance. And when the dose was more than 0.5 Gy, survival fraction exhibited a negative correlation with the dosage. There was no difference between the 0.1 or 0.2 Gy dosage groups and the un-irradiated group in terms of the percentage of cells in M phase. But in the high dosage group (0.3-1.0 Gy), the percentage of cells in M phase was decreased markedly. In addition, the percentage of cells in M phase began to decrease two hours after irradiation. One hour after irradiation, there was no conspicuous activation of Chk2 kinase in 0.1 or 0.2 Gy group, but when the irradiation dose reached 0.3 Gy or higher, Chk2 kinase started to be activated and the activation level showed no significant difference among high dosage groups (0.4, 0.5, 1.0 Gy). Within 1 to 6 h, the DNA DSBs repair efficiency was decreased at 0.2 Gy but increased at 0.5 Gy and 1.0 Gy, which was in line with Chk2 activation. We are led to conclude that the mechanism of HRS/IRR in A549 cell line was probably due to early G(2)/M checkpoint arrest and enhanced DNA DSBs repair. In this regard, Chk2 activation plays a key role in G(2)/M checkpoint activation.
Adenocarcinoma ; genetics ; metabolism ; Cell Line, Tumor ; Checkpoint Kinase 2 ; genetics ; metabolism ; DNA Damage ; genetics ; DNA Repair ; genetics ; Humans ; Lung Neoplasms ; genetics ; metabolism ; Radiation Tolerance ; genetics