Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated anti-aging mechanism in rodents.

Many studies of caloric restriction (CR) in rodents and lower animals indicate that this nutritional manipulation retards aging processes, as evidenced by increased longevity, reduced pathology, and maintenance of physiological function in a more youthful state. The anti-aging effects of CR are believed to relate, at least in part, to changes in energy metabolism. We are attempting to determine whether similar effects occur in response to CR in nonhuman primates. Core (rectal) body temperature decreased progressively with age from 2 to 30 years in rhesus monkeys fed ad lib (controls) and is reduced by approximately 0.5 degrees C in age-matched monkeys subjected to 6 years of a 30% reduction in caloric intake. A short-term (1 month) 30% restriction of 2.5-year-old monkeys lowered subcutaneous body temperature by 1.0 degrees C. Indirect calorimetry showed that 24-hr energy expenditure was reduced by approximately 24% during short-term CR. The temporal association between reduced body temperature and energy expenditure suggests that reductions in body temperature relate to the induction of an energy conservation mechanism during CR. These reductions in body temperature and energy expenditure are consistent with findings in rodent studies in which aging rate was retarded by CR, now strengthening the possibility that CR may exert beneficial effects in primates analogous to those observed in rodents.

Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.

Analysis of differential gene expression by display of 3' end restriction fragments of cDNAs.

We have developed an approach to study changes in gene expression by selective PCR amplification and display of 3' end restriction fragments of double-stranded cDNAs. This method produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on a gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA data bases. Applying this method, we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in this study, as many as 3-4% represented mRNAs that are upregulated, while approximately 2% were down-regulated within 4 hr of activation of Jurkat T cells. These and other results suggest that this approach is suitable for the systematic, expeditious, and nearly exhaustive elucidation of subtle changes in the patterns of gene expression in cells with altered physiologic states.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Restriction-modification systems as genomic parasites in competition for specific sequences.

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Dietary lipids and calorie restriction affect mammary tumor incidence and gene expression in mouse mammary tumor virus/v-Ha-ras transgenic mice.

We have studied the effects of food restriction (FR) and substitution of fish oil (FO; omega 3) for corn oil (CO; omega 6) on breast tumor incidence and survival in mouse mammary tumor virus/v-Ha-ras transgenic (Onco) mice. The diets were as follows: group 1, 5% (wt/wt) CO fed ad libitum (AL); group 2, 5% CO, restricted calories (40% fewer calories than AL; FR); group 3, 20% CO fed AL; and group 4, 20% FO fed AL. After 3 years, 40% of FR Onco (group 2) mice were alive, whereas there were no survivors in the other three groups. Similarly, tumor incidence was reduced to 27% (5 out of 18) in FR animals (group 2), whereas it was 83% (11 out of 13) in group 1 mice, 89% (16 out of 18) in group 3 mice, and 71% (10 out of 14) in group 4 mice. These protective effects of FR on survival and tumor incidence were paralleled by higher expression of the tumor suppressor gene p53 (wild type) and free-radical scavenging enzymes (catalase and superoxide dismutase) in breast tumors. Immunoblotting showed less ras gene product, p21, and increased p53 levels in the tumors of FR mice. In addition, FR decreased RNA levels of c-erbB-2, interleukin 6, and the transgene v-Ha-ras in tumors. In contrast, analysis of hepatic mRNA from tumor-bearing FR mice revealed higher expression of catalase, glutathione peroxidase, and superoxide dismutase. Survival and tumor incidence were not influenced significantly by dietary supplementation with FO in place of CO. Taken together, our studies suggest that moderate restriction of energy intake significantly inhibited the development of mammary tumors and altered expression of cytokines, oncogenes, and free-radical scavenging enzymes.

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Genomic DNA fingerprinting by restriction fragment end labeling.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with [alpha-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.

Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.

Invariance and restriction toward a limited set of self-antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults.

Analysis of the reactivity of IgM with self-antigens in tissues by a quantitative immunoblotting technique showed striking invariance among newborns in the human and in the mouse. The self-reactive repertoire of IgM of adults was also markedly conserved; it comprised most anti-self reactivities that prevailed among neonates. Multivariate analysis confirmed the homogeneity of IgM repertoires of neonates toward self- and non-self-antigens. Multivariate analysis discriminated between newborn and adult repertoires for reactivity with two of five sources of self-proteins and with non-self-antigens. Our observations support the concept that naturally activated B lymphocytes are selected early in development and throughout life for reactivity with a restricted set of self-antigens.