David B. Danner

Abundant alkali-sensitive sites in DNA of human and mouse sperm☆

The DNA of human and mouse sperm cells was analyzed by single-cell microgel electrophoresis, by agarose gel electrophoresis, and by alkaline elution--three techniques that can detect single-strand DNA breaks and/or labile sites. Under these conditions a surprisingly large number of single-strand DNA breaks, approximately 10(6) to 10(7) per genome, were detected in human and mouse sperm but not in human lymphocytes or in mouse bone marrow cells. These breaks were also present in chicken erythrocyte DNA, which is also highly condensed. These breaks were not observed under neutral pH conditions nor under denaturing conditions not involving alkali, suggesting that these sites are alkali-sensitive and do not represent preexisting single-strand breaks. The high frequency of such sites in sperm from healthy mouse and human donors suggests that they represent a functional characteristic of condensed chromatin rather than DNA damage.

DNA damage and rpair with age in individual human lymphocytes

Previous biochemical studies on DNA repair competence and aging have been limited to techniques, such as alkaline elution or nucleoid sedimentation, involving mass cell populations. These techniques provide no information about the distribution of DNA damage and repair among individual cells and are unlikely to detect age-dependent changes affecting a minor fraction of the cell population. We have recently described a microgel electrophoretic assay (Singh et al., 1988) that measures, at the level of the individual cell, single-strand DNA breaks and alkali-sensitive sites. Here, we employ this method to analyze DNA damage and repair in lymphocytes isolated from the peripheral blood of 31 subjects (23 males and 8 females aged 25-91 years) and exposed in vitro to 200 rads of X-irradiation. While basal (pre-irradiation) levels of damage were independent of the age of the donor, an age-dependent increase in DNA damage was observed immediately following irradiation. For all subjects, the mean level of DNA damage was restored to pre-irradiation control levels within 2 h of incubation at 37 degrees C. However, a distribution analysis of DNA damage among cells within each sample indicated the presence of a few highly damaged cells (4-16%) in the 2-h sample, the occurrence of which was significantly more common among aged individuals. These data indicate an age-related decline in DNA repair competence among a small subpopulation of lymphocytes.

Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver

Studies of chromosome loss in inherited cancers, of fusions between proliferating and quiescent cells, and of microinjection of RNA from quiescent cells into proliferation competent cells have all provided evidence for antiproliferative genes in mammalian cells. In this report, we describe a partial cDNA clone isolated on the basis of its preferential hybridization to RNA from normal versus regenerating rat liver. The corresponding mRNA, enriched by hybrid selection, was microinjected into normal human diploid fibroblasts in cell culture, resulting in a 53% decrease in the fraction of nuclei incorporating tritiated thymidine. This mRNA is 2 kb in size and is expressed in eight tissues examined.

Basal DNA damage in individual human lymphocytes with age

A role for DNA damage is central to many theories of aging, but attempts to show an increase in DNA damage with age have yielded contradictory results. However, previous experiments have been of limited sensitivity, only able to examine induced (not basal) damage or pooled (not individual) cells. In this report, we apply a novel technique (Singh et al., 1988) to directly measure basal levels of DNA single-strand breaks and alkali-labile sites in individual human peripheral blood lymphocytes (PBL) obtained from young (less than 60 years) and old (more than 60 years) male donors. This approach shows that while average changes with age are small, changes in certain individuals and in certain cells may be large: the mean increase in damage was only 12%, but the increase in a subpopulation of highly damaged lymphocytes was 5-fold. However, most of this increase was contributed by just 3 of 17 older subjects. Further characterization of these individuals may shed light on the relationship between DNA damage and aging.

Assignment of the human prohibition gene (PHB) to chromosome 17 and identification of a DNA polymorphism

Prohibition is a recently identified antiproliferative protein whose exact role in the cell is under investigation. To determine the human chromosomal location of the prohibition gene (PHB) and whether this site corresponds to that of any suspected tumor suppressor gene, we have analyzed DNA from three sources by hybridization analysis: mouse--human hybrid cell lines, hybrid cell lines containing portions of human chromosomes, and human metaphase chromosomes in situ. All three techniques confirm a location in the region 17q21-q22, a region genetically linked to early-onset human breast cancer. Further analysis will be required to establish the significance of this relationship; Southern hybridizations show a polymorphic EcoRI site that may be useful for this purpose.

Macromolecular synthesis in human diploid fibroblasts: A viral probe examining the effect of in vivo aging

The replication of vesicular stomatitis virus was examined in early-passage skin fibroblast cultures from old and young human donors. The production of virus was analysed by measuring synthesis of viral RNA in actinomycin D-treated cells, and by determining the yield of biologically-active viral progeny by plaque assay. Although no statistically significant differences (P < 0.05) were observed between old and young cultures, our assays were shown to be capable of detecting impaired macromolecular synthesis induced by incorporation of amino acid analogues. These results indicate that macromolecular synthesis does not appear to be significantly altered in cells from older donors.

Changes in fibronectin mRNA splicing with in vitro passage

Senescent human fibroblasts produce larger fibronectin molecules with altered binding properties. To determine if this change could involve alternative splicing of fibronectin precursor mRNA, we developed an approach using reverse transcription and the polymerase chain reaction to study fibronectin mRNA splicing at each of the three alternatively spliced regions. Two of the three regions showed changes with in vitro passage incorporation of the ED-A region increased 8 fold.

Approach to the isolation of antiproliferative genes

Poly(A) RNAs from normal rat liver and senescent human fibroblasts appear to have more antiproliferative activity than RNAs from regenerating rat liver and early passage human fibroblasts. We have screened two rat liver and one human liver library by differential hybridization and isolated four candidate cDNAs for this antiproliferative activity; one is fibronectin and three others do not match to any sequence in the mammalian portion of the GENBANK database. We are currently testing the antiproliferative nature of these cDNAs by microinjection of hybrid-selected RNA, and we describe an alternative strategy for cloning such genes based on construction of a cDNA library in an RNA expression vector.

Isolation and identification of aging-related cDNAs in the mouse

To identify genes whose expression changes as a function of aging, we screened mouse cDNA libraries with cDNAs from mice of different ages. Specifically, whole-mouse cDNA libraries were constructed in lambda gt10 using poly(A) RNA from young (3 month) and old (27 month) C57BL/6J inbred mice and these lambda plaques were hybridized with radioactive cDNAs made from pooled poly(A) RNA from animals 3 or 33 months of age. Five clones were isolated that showed an aging-related pattern of expression and four of these were identified by computerized sequence matching to the GenBank database: MUP2 (a major urinary protein); Q10 of the MHC locus; a cytoskeletal actin gene; and creatine kinase. One gene whose expression increases with aging and is most abundant in spleen remains unidentified. All five cDNAs showed 4-fold to 17-fold changes with aging in their steady-state mRNA levels in at least one tissue.

Aberrant gene expression and aging: Examination of tissue-specific mRNAS in young and old rats

It has been suggested that aberrant gene expression may play a role in aging. To test this possibility, we examined the steady-state mRNA levels for five tissue-specific genes of known function in young (6 month) and old (24 month) rats. Six different tissues from three animals of each age were analyzed using a hybridization assay estimated to be able to detect one mRNA copy per cell. At this level of sensitivity, no aberrant gene expression was seen. The results indicate that these tissue-specific genes retain their fidelity of expression with age.