ABSTRACT
Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.
Subject(s)
Drosophila/metabolism , Fluorescent Dyes/analysis , Kinesins/metabolism , Microtubules/metabolism , Adenosine Triphosphate/metabolism , Animals , Brain/metabolism , DNA Primers/chemistry , DNA, Complementary , Image Processing, Computer-Assisted , Microscopy, Fluorescence/methods , Polymerase Chain Reaction , Protein Biosynthesis , Swine , Transcription, GeneticABSTRACT
Although seminolipid has long been suspected to play an essential role in spermatogenesis because of its uniquely abundant and temporally regulated expression in the spermatocytes, direct experimental evidence has been lacking. We have tested the hypothesis by examining the testis of the UDP-galactose:ceramide galactosyltransferase-deficient mouse, which is incapable of synthesizing seminolipid. Spermatogenesis in homozygous affected males is arrested at the late pachytene stage and the spermatogenic cells degenerate through the apoptotic process. This stage closely follows the phase of rapid seminolipid synthesis in the wild-type mouse. These observations not only provide the first experimental evidence that seminolipid is indeed essential for normal spermatogenesis but also support the broader concept that cell surface glycolipids are important in cellular differentiation and cell-to-cell interaction.
Subject(s)
Galactosyltransferases/genetics , Glycolipids/physiology , Spermatogenesis/physiology , Animals , Base Sequence , DNA Primers , Galactosyltransferases/metabolism , Ganglioside Galactosyltransferase , Male , Mice , Mice, Inbred C57BL , Testis/enzymologyABSTRACT
Mice carrying two t complementary haplotypes (tw5/tw32) are totally sterile. Their spermatozoa have poor motility and fertilize neither zona-intact nor zona-free oocytes, even though they are structurally indistinguishable from control (wild-type) spermatozoa. However, when injected directly into oocytes, these infertile spermatozoa are able to participate in normal development. This suggests that infertility of tw5/tw32 male (spermatozoa) is more likely to be due to poor sperm-oocyte interaction than to genetic incompetence of sperm nuclei.
Subject(s)
Fertilization in Vitro/methods , Infertility, Male , Sperm-Ovum Interactions/physiology , Spermatozoa/physiology , Animals , Female , Male , MiceABSTRACT
Day-night variation of the preprosomatostatin mRNA level in the suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, was determined in rats maintained under light-dark cycles by semiquantitative Northern blot hybridization of micropunched tissues. Preprosomatostatin mRNA abundance in the SCN showed significant variation during a day with a peak level at around light on and a trough at around 4 h after light off. This time course was essentially identical with that previously found in blinded rats, indicating that environmental light did not produce an immediate effect on the preprosomatostatin mRNA level in the SCN. Moreover, this rhythm in the preprosomatostatin mRNA appeared specific to the SCN, since levels in the cortex and anterior hypothalamus did not oscillate even under light-dark conditions.
