An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues.

Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity.

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

Oncornavirus lytic activity in the serum of gibbon apes.

Fresh blood serum from normal gibbon apes (Hylobates lar) contained heat-sensitive lytic activity for various mammalian oncornaviruses. Lytic activity quantitatively similar to that in gibbon serum was demonstrated in serum from three other primate species, including man; it was demonstrated to be low or absent in lower mammalian species with the exception of domestic cats, which had intermediate levels of serum lytic activity. Gibbons that acquired infectious gibbon ape leukemia virus, either naturally by exposure to a virus-shedding ape or experimentally by deliberate virus inoculation, had the same levels of serum lytic activity as did unexposed gibbons that had no detectable antibodies to gibbon ape leukemia virus. A leukemic-viremic gibbon had low or absent serum oncornavirus lytic activity. These results indicated that serum lytic activity does not necessarily protect against infection by oncornaviruses, although it may limit virus replication and/or dissemination.

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Rat synapsin 1 promoter mediated transgene expression in testicular cell types.

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.

Using MT(-/-) mice to study metallothionein and oxidative stress.

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.

Analysis and modulation of DNA repair in aging.

Nearly 40 years ago it was proposed that accumulation of mutations or increased levels of DNA damage might contribute to aging processes. Despite several correlative studies in this area, the answer as to whether genomic integrity contributes to aging has remained illusive. More recently it has been hypothesized that decreased mitochondrial DNA integrity plays a role in aging. To begin to test these hypotheses more directly, we are developing transgenic mouse and cell culture model systems. For example, transgenic mice overexpressing the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) have been made and have a reduced spontaneous frequency of hepatocellular carcinoma. A lifespan study using the MGMT transgenic mice is in progress in an effort to determine whether cancer impacts on the median or maximal lifespan of a species. Second, a quantitative PCR technique is being used to measure mitochondrial DNA damage in mitotic and post-mitotic cells to determine if the level of damage and/or repair is different based on mitotic status. Finally, mice deficient in metallothionein-I and -II are being used in an effort to determine if the subcellular distribution of metals impact on oxidative damage with increased age.

Hypothermic perfusion of rabbit kidneys with solutions containing gelatin polypeptides.

Rabbit kidneys were perfused with a solution of extracellular electrolyte composition, made hypertonic with glucose and containing the gelatin polypeptide preparation Haemaccel (Hoechst) as the only colloid. Perfusions were carried out at 5 and 10 C for 19 hr, and function was tested by autografting. All of the kidneys perfused at the higher temperature showed immediate life-sustaining function after transplantation and contralateral nephrectomy, whereas only one graft of five perfused at the lower temperature showed any function. The suitability of the Haemaccel solution as a vehicle for introducing the cryoprotective agent glycerol was tested by perfusing kidneys for 4 hr with a solutiont containing 2% glycerol; the function of these organs was similar to that of kidneys transplanted without perfusion. Ultrastructural examination of kidneys perfused for 24 hr at 10 C showed excellent structural preservation, but measurements of water and ion contents and the penetration of marker molecules in nonmetabolizing kidneys showed 2.8% Haemaccel to be somewhat less effective than 6% bovine serum albumin in stabilizing these values. The Haemaccel perfusate is considered to be highly suitable for the introduction and removal of cryoprotective agents, and the results of hypothermic preservation by continuous perfusion are encouraging.

Second 46,XX male with MLS syndrome.

We report on a second 46,XX male with microphthalmia with linear skin defects (MLS) syndrome. In addition to microphthalmia and linear skin streaks, he had a secundum ASD, hypospadias with chordee, anal fistula, and agenesis of corpus callosum with colpocephaly. Biopsy of a linear streak showed smooth muscle hamartomata rather than the presumed dermal aplasia. Detailed ophthalmologic examination did not show retinal lacunae typical of Aicardi syndrome. DNA studies with distal Xp specific probes indicated a deletion in one X chromosome and fluorescence in situ hybridization (FISH) studies with X- and Y-specific probes demonstrated the presence of a derivative X chromosome from an X;Y translocation.

Short-limb dwarfism and hypertrophic cardiomyopathy in a patient with paternal isodisomy 14: 45,XY,idic(14)(p11).

Uniparental disomy (UPD) has been shown to result in specific disorders either due to imprinting and/or homozygosity of mutant alleles. Here we present the findings in a child with paternal UPD14. Ultrasound evaluation was performed at 30 weeks of gestation because of abnormally large uterine size. Pertinent ultrasound findings included polyhydramnios, short limbs, abnormal position of hands, small thorax, and nonvisualization of the fetal stomach. Post-natally the infant was found to have a low birth weight, short birth length, contractures, short limbs, and a small thorax with upslanting ribs. Assisted ventilation and gastrostomy were required. At age 6 months, the infant required hospitalization for hypertrophic cardiomyopathy which responded to Atenolol. Initial cytogenetic studies demonstrated an apparently balanced de novo Robertsonian translocation involving chromosomes 14 and a karyotype designation of 45,XY,t(14q14q). No indication of mosaicism for trisomy 14 was observed in metaphase spreads prepared from peripheral blood lymphocytes or skin-derived fibroblasts. C-band and fluorescence in situ hybridization results demonstrated that the chromosome was dicentric. DNA analyses showed paternal uniparental isodisomy for chromosome 14. Based on the cytogenetic and DNA results a final karyotype designation of 45,XY,idic(14)(p11) was assigned to this infant with paternal isodisomy of chromosome 14.

Health span and life span in transgenic mice with modulated DNA repair.

One way to better understand the contribution of DNA repair, DNA damage, and mutagenesis in aging would be to enhance DNA repair activity, lower DNA damage, and lower mutagenesis. Because the repair protein O6-methylguanine-DNA methyltransferase (MGMT) acts alone and stoichiometrically, the human MGMT (hMGMT) cDNA was selected to test the feasibility of enhancing DNA repair activity in transgenic mice. MGMT activity is largely responsible for ameliorating the deleterious effects of O6-methylguanine (O6mG) lesions in DNA in a direct reversal mechanism. A transgene was constructed consisting of a portion of the human transferrin (TF) promoter and hMGMT cDNA such that hMGMT is expressed in transgenic mouse brain and liver. Expression of hMGMT was associated with a significant reduction in the occurrence of an age-related hepatocellular carcinoma in male mice at 15 months of age. Longitudinal and cross-sectional studies were initiated to determine whether the reduced incidence of hepatocellular carcinoma would impact median or maximum life span. The cross-sectional study performed on 15-month-old male animals confirmed the reduced occurrence of spontaneous hepatocellular carcinoma. At 30 months of age, however, the occurrence of hepatocellular carcinoma in at least one transgenic line was similar to that for nontransgenic animals. The longitudinal study is ongoing; however, at present no significant differences in life span have been detected. Tissues expressing the MGMT transgene also displayed greater resistance to alkylation-induced tumor formation. These results suggest that transgenes can be used to direct enhanced DNA repair gene expression and that enhanced expression can protect animals from certain spontaneous and induced tumors.

Application of exogenously regulatable promoter systems to transgenic models for the study of aging.

Transgenic mouse and gene knockout technologies offer powerful tools for dissecting the roles of specific genes in the process of aging. Tke interpretation of the results of such studies is limited, however, by the fact that the gene of interest of over- or underexpressed throughout the life span of the animal model. Among other problems, this situation makes it difficult to separate the effects that a specific gene has an embryological development from those that it may exert on the subsequent maturation and aging of the animal. It is also not possible with these methods alone to alter the expression of genes in an age-dependent fashion and to assess the effects of these alterations on the aging process. This capacity would be of particular interest in studying genes which are thought to have a role in regulating physiological homeostasis. Because they offer the opportunity to activate or render inactive the expression of genes at will, exogenously regulatable promoter systems, particularly when used in combination with traditional transgenic or gene knockout approaches, provide a new and potentially very powerful tool for studying the effect of selected genes on aging. This review discusses the merits and limitations of the application of either the tetracycline-regulatable promoter system, the RU 486-inducible promoter system, or the ecdysone-inducible promoter system to exogenously regulate the expression of a transcriptionally linked gene and to thus assess the effect of that gene on aging.

YY1 and Sp1 transcription factors bind the human transferrin gene in an age-related manner.

The iron-binding protein transferrin has major roles in transporting, delivering, and sequestering ferric ions acquired by body tissues. Yet, during aging, serum transferrin levels decrease in humans. Likewise, in transgenic mice carrying chimeric human transferrin transgenes, liver expression of transferrin transgenes decreases with age. The aging regulation is due to decreased gene transcription. Electrophoretic mobility shift assays and antibody-recognition have revealed the binding of 5' regulatory elements of the human transferrin gene by three YY1 proteins, called YY1, YY1-a, and YY1-b, and an Sp1-a transcription factor. An age-related increase in YY1-a and YY1-b binding activities and a decrease in Sp1-like binding activity were shown. Since Sp1 is a positive transcription factor and YY1 can be a negative transcription factor, the alterations in their binding with age could cause the decreased transcription of the human transferrin transgene, and also the age-related decreased serum transferrin levels in humans.

A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide.

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver.

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.

Expression of the DNA repair gene XRCC1 in baboon tissues.

XRCC1 is a DNA repair gene involved in rejoining DNA strand-breaks. We used baboon as an animal model to determine the levels of XRCC1 gene expression in different tissues. Baboons were selected because they are evolutionarily closely related to humans. A single 2.2 kb transcript was detected in all tissues tested by northern blot analysis, with variations in levels of expression among tissues. The expression levels of XRCC1 were measured by quantitative RNase protection assays. XRCC1 mRNA levels were significantly higher in testis than those in other tissues. A mean value of 24.6 x 10(5) XRCC1 transcripts per micrograms DNA was found in testis, while 10.5 x 10(5) in ovary, 9.8 x 10(5) in brain, 8.5 x 10(5) in liver, 6.8 x 10(5) in kidney, 6.5 x 10(5) in heart, 6.4 x 10(5) in lymph nodes, 6.0 x 10(5) in lung and 4.9 x 10(5) in spleen were found.

Characterization of O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in Xiphophorus fishes.

We utilized a custom-synthesized double-strand oligonucleotide containing a single O(6)-methylguanine (O(6)-MG) residue within a restriction endonuclease recognition site to determine O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in various tissue extracts prepared from Xiphophorus fish. The results suggest Xiphophorus fish O(6)-MGMT activity has many of the same characteristics as Escherichia coli and mammalian O(6)-MGMT's including rapid reaction kinetics consistent with stoichiometric removal of methyl groups, but exhibits a temperature optimum of 23 degrees C. Results from protein extract activity assays indicate O(6)-MGMT activity patterns among four Xiphophorus tissues followed the order: brain> or =testes>gill> or =liver. In mammals, O(6)-MGMT activity is high in liver, while activity in brain is minimal (i.e. approximately 9% of liver); however, we report that in the Xiphophorus fishes examined, brain tissue extracts exhibited much higher (approximately six-fold) O(6)-MGMT activity levels than liver. Comparison of O(6)-MGMT activity between Xiphophorus species employed in tumor induction experiments did not indicate significant differences in ability to clear the pre-mutagenic O(6)-MG from the oligonucleotide substrate.

Relative base excision repair in Xiphophorus fish tissue extracts.

To begin characterizing DNA repair capability among Xiphophorus species, we adapted oligonucleotide-based DNA repair assays to extracts of fish tissues. Here, we report the initial results of relative base excision repair (BER) capability among 3 inbred Xiphophorus fish lines representing 2 species (X. maculatus and X. couchianus), and interspecies hybrid F(1) animals produced by crossing them. Overall, data from uracil- N-glycosylase (UNG)-initiated BER assay (UNG-BER) indicate that brain tissue extracts generally possess higher BER activity than do gill and liver extracts. UNG-BER activities in gill and liver extracts were similar. The BER activity in the tissues of F(1) interspecies hybrids followed the activity pattern of the X. couchianus parent in gill and liver extracts, was similar to the X. maculatus parent in brain extracts, but exhibited a reduced repair capacity in gill tissue extracts compared with either parent. We discuss the use of oligonucleotide-based DNA repair assays to elucidate the role that inheritance of DNA repair potential may play in susceptibility to disease and tumorigenesis in the intact organism.