Dose-response of an induction of a germline mutation was studied at a hypervariable mouse minisatellite locus, Ms6hm, which consists of tandem repeats of a sequence motif GGGCA. Male C3H/HeN mice were exposed to various doses of 60Co gamma-ray and mated with unirradiated C57BL/6N female mice. Matings were done at various time after irradiation to assess the effects of radiation on spermatozoa, spermatids and spermatogonia. DNA samples of F1 offsprings were analysed by Southern blotting for the repeat length mutation at the Ms6hm locus. The mutation frequency per gamete of the paternal allele was 9.1% for the unirradiated control group. The spermatids stage was most sensitive to radiation and a statistically significant dose-response was observed. The mutation frequency of the paternal allele in F1 mice increased to 22% for 1 Gy, 28% for 2 Gy, and 28% for 3 Gy. The spermatogonia stage was less sensitive to radiation, and the mutation frequencies of the paternal allele were 14% for 2 Gy, and 16% for 3 Gy. The spermatozoa stage germ cells were also less sensitive and the frequency of mutation of the paternal allele increased to 14% for 3 Gy. However, these increases were statistically not significant. Possible mechanisms of radiation induction of germline mutation at the hypervariable minisatellite locus will be discussed.

Analysis was made on the genomic structure, functions, and expression of the mouse ELP gene, which codes for the embryonal long terminal repeat binding protein. Extensive screening of the cDNA library of embryonal carcinoma cells (EC cells) identified four isoforms of ELP: ELP1 (the original ELP isolate), ELP2, ELP3, and Ad4BP/SF1. Analysis of the genomic sequences revealed that these ELP isoforms were generated by alternative promoter usage and differential splicing. The mRNAs of isoforms initiated at four transcription start sites distributed on three exons. Sequence analysis of the four isoforms identified three polypeptides. The N-terminal portion of ELP1 and ELP2 was longer than ELP3, and Ad4BP/SF1 by 77 aa. The DNA-binding domain and region II were shared by all four isoforms. The C-terminal portion shared by ELP2, ELP3, and Ad4BP/SF1 was 131 aa in length, and that specific to ELP1 was 57 aa in length. The ELP3 and Ad4BP/SF1 isoforms were identical for the coding sequence, but the two differed at the 5' noncoding region. Region II and III domains of nuclear receptors were thought to be involved in ligand-binding and transcriptional activation. ELP1, which lacked region III, functioned as a repressor. The isoforms carrying intact region II and region III functioned as transactivators. Expression of the four isoforms was studied in mouse tissues and in tissue culture cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Complex patterns of expression of these isoforms were observed in various tissues. All four ELP isoforms were expressed only in EC cells.

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4؉ T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27 Kip1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.