ABSTRACT
Long interspersed elements (LINEs) replicate by target primed reverse transcription (TPRT). Insertion involves two half reactions. Each half reaction involves DNA cleavage followed by DNA synthesis. The linker region, located just beyond the reverse transcriptase in the LINE open reading frame, contains a set of predicted helices that may form an α-finger, followed by a gag-like zinc-knuckle. Point mutations of moderately conserved amino-acid residues in the presumptive α-finger severely impair the DNA endonuclease and reverse transcriptase activities of the integration reaction during both half reactions. Mutations in the gag-like zinc-knuckle also impair DNA cleavage and DNA synthesis in some instances. Mutations in core residues that presumably disrupt the protein structure of the presumptive α-finger and the gag-like zinc-knuckle lead to a promiscuous DNA endonuclease and protein-nucleic acid complexes that get stuck in the well during analysis. The linker region appears to function as a protein, DNA, and RNA conformational switching area. The linker is used to properly position nucleic acid substrates into the active sites of the reverse transcriptase and of the DNA endonuclease.
Subject(s)
DNA/chemistry , DNA/metabolism , Long Interspersed Nucleotide Elements/physiology , Amino Acid Motifs , Binding Sites , Conserved Sequence , DNA/biosynthesis , DNA Cleavage , Deoxyribonuclease I/metabolism , Insect Proteins , Point Mutation , Polymerization , Protein Structure, Secondary , Protein Structure, Tertiary , RNA-Directed DNA Polymerase/metabolismABSTRACT
Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented.
Subject(s)
DNA Restriction Enzymes/genetics , DNA, Cruciform/genetics , Long Interspersed Nucleotide Elements , RNA, Messenger/genetics , RNA-Directed DNA Polymerase/genetics , Reverse Transcription , Animals , Bombyx/genetics , Bombyx/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Cleavage , DNA Primers/genetics , DNA Primers/metabolism , DNA Restriction Enzymes/metabolism , DNA, Cruciform/chemistry , DNA, Cruciform/metabolism , Holliday Junction Resolvases/genetics , Holliday Junction Resolvases/metabolism , Nucleic Acid Conformation , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase/metabolismABSTRACT
Non-LTR retrotransposons are an important class of mobile elements that insert into host DNA by target-primed reverse transcription (TPRT). Non-LTR retrotransposons must bind to their mRNA, recognize and cleave their target DNA, and perform TPRT at the site of DNA cleavage. As DNA binding and cleavage are such central parts of the integration reaction, a better understanding of the endonuclease encoded by non-LTR retrotransposons is needed. This paper explores the R2 endonuclease domain from Bombyx mori using in vitro studies and in silico modeling. Mutations in conserved sequences located across the putative PD-(D/E)XK endonuclease domain reduced DNA cleavage, DNA binding and TPRT. A mutation at the beginning of the first α-helix of the modeled endonuclease obliterated DNA cleavage and greatly reduced DNA binding. It also reduced TPRT when tested on pre-cleaved DNA substrates. The catalytic K was located to a non-canonical position within the second α-helix. A mutation located after the fourth ß-strand reduced DNA binding and cleavage. The motifs that showed impaired activity form an extensive basic region. The R2 biochemical and structural data are compared and contrasted with that of two other well characterized PD-(D/E)XK endonucleases, restriction endonucleases and archaeal Holliday junction resolvases.
Subject(s)
Endodeoxyribonucleases/chemistry , Retroelements , Amino Acid Sequence , Animals , Bombyx/enzymology , Conserved Sequence , DNA/metabolism , DNA Cleavage , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Models, Molecular , Mutation , Protein Structure, Secondary , Reverse Transcription , Sequence AlignmentABSTRACT
Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of ?-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (PPin vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility.
ABSTRACT
Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = -0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = -0.49, P<0.006 and r = -0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1-3 vs. Bull 4-5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.
Subject(s)
Apoptosis , Fertility , Spermatozoa/pathology , Spermatozoa/physiology , Animals , Cattle , DNA Damage , Flow Cytometry , Male , Phosphatidylserines/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Reproduction , Sperm Motility , bcl-2-Associated X Protein/metabolismABSTRACT
BACKGROUND: MicroRNAs are small non-coding RNAs that regulate gene expression and thus play important roles in mammalian development. However, the comprehensive lists of microRNAs, as well as, molecular mechanisms by which microRNAs regulate gene expression during gamete and embryo development are poorly defined. The objectives of this study were to determine microRNAs in bull sperm and predict their functions. METHODS: To accomplish our objectives we isolated miRNAs from sperm of high and low fertility bulls, conducted microRNA microarray experiments and validated expression of a panel of microRNAs using real time RT-PCR. Bioinformatic approaches were carried out to identify regulated targets. RESULTS: We demonstrated that an abundance of microRNAs were present in bovine spermatozoa, however, only seven were differentially expressed; hsa-aga-3155, -8197, -6727, -11796, -14189, -6125, -13659. The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa. CONCLUSION: Identification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development.
Subject(s)
Cattle/genetics , Fertility/genetics , MicroRNAs/genetics , Animals , Male , MicroRNAs/metabolism , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Spermatozoa/physiologyABSTRACT
Fertilization of an egg by a spermatozoon sets the stage for mammalian development. Viable sperm are a prerequisite for successful fertilization and beyond. Spermatozoa have a unique cell structure where haploid genomic DNA is located in a tiny cytoplasmic space in the head, mitochondria in the midpiece and then the tail, all enclosed by several layers of membrane. Proteins in sperm play vital roles in motility, capacitation, fertilization, egg activation and embryo development. Molecular defects in these proteins are associated with low fertility or in some cases, infertility. This review will first summarize genesis, molecular anatomy and physiology of spermatozoa, fertilization, embryogenesis and then those proteins playing important roles in various aspects of sperm physiology.