Tetsuya Moriuchi

Localizationin situ ofc-myc mRNA andc-myc protein in adult mouse testis

SummaryIt has been suggested that c-myc, one of the proto-oncogenes, plays a role in normal somatic cell proliferation and differentiation. To define whether c-myc is only expressed during somatic cell division or is also expressed during meiotic cell division, the production of c-myc mRNA and protein were investigated in the mouse testis by usingin situ hybridization with non-radioactive DNA probes and enzyme immunohistochemistry respectively. Forin situ hybridization, T-T dimerized DNA probes were used and DNAs hybridizedin situ were detected immunohistochemically using specific antibody against T-T dimer. The results indicate that c-myc mRNA and protein are expressed in a cell-cycle-dependent manner only in spermatogonia and not in spermatocytes and spermatids.

Rat Thy-1 antigen has a hydrophobic segment at the carboxyl terminus.

We have isolated a cDNA clone coding for rat Thy‐1 antigen from a rat thymocyte cDNA library. The 549 base pairs insert includes the complete coding region of a mature Thy‐1 polypeptide of 142 amino acids, 31 amino acids longer than the previously reported glycoprotein sequence. These 31 amino acids contain an extremely hydrophobic region of 26 uncharged amino acids which apparently represent the transmembrane segment that allows for integration within the membrane. The presence of this additional protein segment will solve most of the enigmas regarding the properties of the C‐terminal region of Thy‐1 antigen.

Molecular Analysis of Abnormal Satellite I Dna from a Buf/Mna Rat Thymoma

BUF/Mna rats develop spontaneous thymomas in an autosomal dominant manner. We constructed recombinant plasmid library of 90 and 185 base-paired (bp) satellite I DNA fragments isolated from BUF/Mna rat thymoma DNA. Four unusual clones containing 93, 95, 95, and 173 bp inserts were isolated by colony hybridization with Wistar rat satellite I DNA. Nucleotide sequence analysis of the inserts of the 4 clones revealed abnormal sequence organization and unusual subunit structure of the rat satellite I DNA. Sequence comparisons between normal and abnormal satellite I DNA suggested that the unusual subunit structure could be generated by the change of the Hinf I recognition sequence to an Eco RI cleavage site, in addition to random deletions, insertions and base substitutions. The heptanucleotide sequence TGGGAAC, which is strictly conserved in normal subunits, was completely lost in all these clones. Southern blot hybridization revealed the amplification of abnormal satellite I DNA in BUF/Mna rat thymomas.

Non‐Radioactive cDNA Probes for in situ Localization of mRNA

DNA labeled with non‐radioactive markers has been utilized for detection of specific DNA or RNA either on filters or in cells and tissues. The presence of protruding markers on the probe DNA has been considered to be a cause for loss of sensitivity and specificity of hybridization. As a non‐protruding marker, we introduced the use of T‐T dimer, which can be generated easily and is a potent hapten. As a marker for DNA, T‐T dimer in DNA was generated by UV irradiation. The T‐T dimerized DNA (T‐T DNA) was then hybridized with complementary DNA or mRNA either on a nitrocellulose filter or in cells. Then the hybridized T‐T DNA was detected immunohistochemically using rabbit anti‐T‐T DNA and peroxidase‐labeled goat anti‐rabbit IgG. The use of T‐T as marker offers several advantages over other markers; it appears not to interfere with hybridization efficiency, is simple to make and can be detected with high sensitivity.