ABSTRACT
The formation of the blood-testis barrier (BTB) is defined as occurring with the first appearance of spermatocytes at around puberty and is vital for normal spermatogenesis. This barrier between two adjacent Sertoli cells (SCs) consists of a cell junctional protein complex, which includes tight junctions (TJs), adherens junctions, and gap junctions. In many mammalian species, BTB composition has already been investigated, whereas little is known about the equine BTB. In the present study, immunohistochemistry and qualitative Western Blot analysis were used to assess the expression and distribution patterns of the junctional proteins claudin-11 (TJ), zonula occludens-1 (TJ associated), N-cadherin (adherens junctions), and connexin 43 (gap junctions) in equine testes during tubular development and in testes of stallions exhibiting unilateral cryptorchidism. Therefore, testes of 21 warmblood stallions (aged 12 months-11 years) were obtained during routine surgical castration. In the normal adult equine testis, the junctional proteins are localized at the basolateral region of the seminiferous tubules forming a circumferential seal corresponding to the known BTB localization. N-cadherin is additionally expressed along the lateral SC surface. In immature seminiferous cords still lacking a lumen, a diffuse distribution pattern of the junctional proteins throughout the SC cytoplasm is visible. As lumen formation advances, the immunolocalization shifts progressively toward the basolateral SC membranes. Additionally, apoptotic germ cells were detected and quantified in prepubertal stallions using terminal deoxynucleotidyl transferase dUTP nick end labeling assay and correlated with junctional protein localization. In the retained testis of cryptorchid stallions, which exhibit an aberrant testicular morphology, a deviating expression of the junctional proteins is visible. The present data show for the first time that (1) the equine SC junctional complex contains claudin-11, zonula occludens-1, N-Cadherin, and connexin 43, as already described for men or mice, and that (2) different distribution patterns of these proteins exist during testicular development in the context of lumen formation (lumen scores: 1-7) and in retained testes of unilateral cryptorchid stallions.
Subject(s)
Blood-Testis Barrier/growth & development , Horses/physiology , Animals , Blood-Testis Barrier/metabolism , Blotting, Western , Cadherins/metabolism , Claudins/metabolism , Connexin 43/metabolism , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Nick-End Labeling , Male , Sexual Maturation , Testis/growth & developmentABSTRACT
As DNA sequencing is performed more and more in a mass-production-like manner, efficient quality control measures become increasingly important for process control, but so also does the ability to compare different methods and projects. One of the fundamental quality measures in sequencing projects is the position-specific error probability at all bases in each individual sequence. Accurate prediction of base-specific error rates from "raw" sequence data would allow immediate quality control as well as benchmarking different methods and projects while avoiding the inefficiencies and time delays associated with resequencing and assessments after "finishing" a sequence. The program PHRED provides base-specific quality scores that are logarythmically related to error probabilities. This study assessed the accuracy of PHRED's error-rate prediction by analyzing sequencing projects from six different large-scale sequencing laboratories. All projects used four-color fluorescent sequencing, but the sequencing methods used varied widely between the different projects. The results indicate that the error-rate predictions such as those given by PHRED can be highly accurate for a large variety of different sequencing methods as well as over a wide range of sequence quality.
Subject(s)
Base Sequence , Sequence Analysis, DNA/methods , Sequence Analysis, DNA/statistics & numerical data , Chromosomes, Bacterial , Cosmids , Frameshift Mutation , Reproducibility of Results , Sequence Alignment , Sequence Homology, Nucleic Acid , SoftwareABSTRACT
The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.
Subject(s)
DNA, Bacterial/isolation & purification , Genome, Bacterial , Mycobacterium leprae/genetics , Sequence Analysis, DNA , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Computing Methodologies , Cosmids/isolation & purification , Molecular Sequence Data , Multienzyme Complexes/genetics , Mycobacterium tuberculosis/genetics , Open Reading Frames/genetics , Operon/genetics , Pseudogenes , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidSubject(s)
Base Sequence , DNA , Biotin , Digoxigenin , Electrophoresis, Polyacrylamide Gel/instrumentation , Electrophoresis, Polyacrylamide Gel/methods , Immunoenzyme Techniques , Indicators and Reagents , Luminescent Measurements , Mathematics , Models, Theoretical , Molecular Sequence Data , OligodeoxyribonucleotidesABSTRACT
Methods for the nonradioactive chemical sequencing of DNA are described. A biotin marker molecule, attached chemically to an oligonucleotide primer or enzymatically in an endfilling reaction of restriction enzyme sites, is stable during the base-specific chemical modification and strand scission reactions. Following fragment separation by direct blotting electrophoresis, the membrane bound sequence pattern can be visualized by a streptavidin-bridged enzymatic color reaction. The biotin labeling is also applicable for DNA sequencing by random degradation of phosphothioates, thus showing to be a universal label for nonradioactive DNA sequencing.
Subject(s)
Base Sequence , Biotin , DNA , Bacterial Proteins , Chemical Phenomena , Chemistry , Colorimetry , Electrophoresis , Epoxy Compounds , Organothiophosphates , Propanols , StreptavidinABSTRACT
We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.
