Vertebrate genomes are characterized with CpG deficiency, particularly for GC-poor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5' T to 5' A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5' Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected (obs/exp) values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes (except sea urchin) but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5' T and 5' A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.
AT Rich Sequence ; Animals ; CpG Islands ; genetics ; DNA Methylation ; GC Rich Sequence ; Gene Frequency ; Genome ; Genomics ; methods ; Humans ; Invertebrates ; genetics ; Isochores ; genetics ; Mutation ; Vertebrates ; genetics

The energy of interaction between complementary nucleotides in promoter sequences of E. coli was calculated and visualized. The graphic method for presentation of energy properties of promoter sequences was elaborated on. Data obtained indicated that energy distribution through the length of promoter sequence results in picture with minima at -35, -8 and +7 regions corresponding to areas with elevated AT (adenine-thymine) content. The most important difference from the random sequences area is related to -8. Four promoter groups and their energy properties were revealed. The promoters with minimal and maximal energy of interaction between complementary nucleotides have low strengths, the strongest promoters correspond to promoter clusters characterized by intermediate energy values.
AT Rich Sequence ; genetics ; Base Sequence ; Chromosome Mapping ; methods ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA ; methods ; Transcription, Genetic ; genetics

OBJECTIVETo identify whether GC-box (-348 to -338) in human insulin gene promoter is a key cis-acting element.

METHODSHuman insulin gene promoter was sub-cloned into secreted alkaline phosphatase (SEAP) reporter plasmid. The deletion and mutation of GC-box in insulin gene promoter was performed. The activity of human insulin gene promoter was determined by evaluating the activity of SEAP in the supernatant of cell culture after the reporter plasmids were transfected in beta cell line betaTC3.

RESULTDeletion and mutation of GC box in human insulin gene promoter did not result in significant changes of the activity of the promoter in betaTC3.

CONCLUSIONThe GC-box is not a key cis-acting element in human insulin gene promoter.

Alkaline Phosphatase ; genetics ; metabolism ; Base Sequence ; Cell Line ; Enhancer Elements, Genetic ; GC Rich Sequence ; Gene Expression Regulation ; Humans ; Insulin ; genetics ; Promoter Regions, Genetic ; Recombinant Fusion Proteins ; genetics ; metabolism ; Sequence Deletion ; Transcription, Genetic ; Transfection

The leucine-rich repeat kinase2 (LRRK2) has been identified to be the gene causing autosomal dominant inherited Parkinson's disease(PD)8. The clinical features of this type of PD are similar to those of idiopathic PD, but the pathological changes are diverse. The mutation types and frequencies of the LRRK2 distribute unevenly in different populations. LRRK2 is a large complex protein with multiple functions and expresses widely in human body. Sequence alignment shows that LRRK2 might be a multiple function kinase for substrate phosphorylation and might also act as a scaffolding protein. Further study on the physiological function and pathogenic mechanism of LRRK2 will help to find out the possible pathogenesis and new treatment for PD.
Animals ; Continental Population Groups ; genetics ; Humans ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ; Mutation ; Parkinson Disease ; genetics ; pathology ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Sequence Alignment

OBJECTIVETo systematically explore the occurrence of a novel type of chromosome translocation in human sperm samples.

METHODSSpecific translocation junction fragments were quantified using nested and/or multi-nested PCR in sperm DNA derived from 28 oligospermic patients and 32 normal controls.

RESULTSt(11;22) was detected in 49 samples. At least 4 samples were found to have t(1;22) (p21.2;q11.2), t(17;22) (q11;q11) or t(X;22) (q27;q11). The mutation rate seemed to be associated not with age or semen volume, but with sperm concentration (r = -0.389, P < 0.05) and motility (r = -0.397, P < 0.05). Correlation was not found between homology of palindromic sequences and mutation rate.

CONCLUSIONPalindromic sequence mediated chromosome translocation is common in human sperm, and associated with sperm concentration and motility. Measurement of such mutations may provide a molecular-level reference for assessing sperm quality.

AT Rich Sequence ; Adult ; Base Sequence ; Chromosomes, Human, Pair 11 ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 22 ; Chromosomes, Human, X ; Humans ; Male ; Middle Aged ; Mutation ; Oligospermia ; genetics ; Spermatozoa ; metabolism ; Translocation, Genetic

OBJECTIVES: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. METHODS: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (connective tissue growth factor/cysteine-rich 61/nephroblastoma-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. RESULTS: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. CONCLUSIONS: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.
Animals ; Cell Enlargement* ; Cell Size* ; Cysteine-Rich Protein 61 ; Cytoplasm ; Female ; Gene Expression ; Genes, vif ; Granulosa Cells ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Mice* ; Oligonucleotide Array Sequence Analysis ; Oocytes ; Ovary ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Theca Cells

OBJECTIVE: To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC. METHODS: Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively. RESULTS: Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05). CONCLUSION TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.
Base Sequence ; Cell Line ; Cold Temperature ; Epithelial Cells ; cytology ; metabolism ; Exocytosis ; physiology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Mucin 5AC ; metabolism ; Mutation ; Myristoylated Alanine-Rich C Kinase Substrate ; Phosphorylation ; TRPM Cation Channels ; metabolism ; Trachea ; cytology ; metabolism

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.
Amino Acid Sequence ; Animals ; Benzeneacetamides ; chemical synthesis ; pharmacology ; Cattle ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dental Implants ; Enzyme-Linked Immunosorbent Assay ; Epithelial Attachment ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Piperidones ; chemical synthesis ; pharmacology ; Platelet-Rich Plasma ; Receptors, Thrombin ; Surface Properties ; Titanium ; chemistry