Telomere length as a quantitative trait: genome-wide survey and genetic mapping of telomere length-control genes in yeast

Telomere length-variation in deletion strains of Saccharomyces cerevisiae was used to identify genes and pathways that regulate telomere length. We found 72 genes that when deleted confer short telomeres, and 80 genes that confer long telomeres relative to those of wild-type yeast. Among identified genes, 88 have not been previously implicated in telomere length control. Genes that regulate telomere length span a variety of functions that can be broadly separated into telomerase-dependent and telomerase-independent pathways. We also found 39 genes that have an important role in telomere maintenance or cell proliferation in the absence of telomerase, including genes that participate in deoxyribonucleotide biosynthesis, sister chromatid cohesion, and vacuolar protein sorting. Given the large number of loci identified, we investigated telomere lengths in 13 wild yeast strains and found substantial natural variation in telomere length among the isolates. Furthermore, we crossed a wild isolate to a laboratory strain and analyzed telomere length in 122 progeny. Genome-wide linkage analysis among these segregants revealed two loci that account for 30%–35% of telomere length-variation between the strains. These findings support a general model of telomere length-variation in outbred populations that results from polymorphisms at a large number of loci. Furthermore, our results laid the foundation for studying genetic determinants of telomere length-variation and their roles in human disease.     

Wild-derived inbred mouse strains have short telomeres

Telomere length and telomerase activity directly affect the replicative capacity of primary human cells. Some have suggested that telomere length influences organismal lifespan. We compared telomere length distributions in a number of inbred and outbred established mouse strains with those of strains recently derived from wild mice. Telomere length was considerably shorter in wild-derived strains than in the established strains. We found no correlation of telomere length with lifespan, even among closely related inbred mouse strains. Thus, while telomere length plays a role in cellular lifespan in cultured human cells, it is not a major factor in determining organismal lifespan.     

Inbreeding is associated with shorter early-life telomere length in a wild passerine

Inbreeding can have negative effects on survival and reproduction, which may be of conservation concern in small and isolated populations. However, the physiological mechanisms underlying inbreeding depression are not well-known. The length of telomeres, the DNA sequences protecting chromosome ends, has been associated with health or fitness in several species. We investigated effects of inbreeding on early-life telomere length in two small island populations of wild house sparrows (Passer domesticus) known to be affected by inbreeding depression. Using genomic measures of inbreeding we found that inbred nestling house sparrows (n?=?371) have significantly shorter telomeres. Using pedigree-based estimates of inbreeding we found a tendency for inbred nestling house sparrows to have shorter telomeres (n?=?1195). This negative effect of inbreeding on telomere length may have been complemented by a heterosis effect resulting in longer telomeres in individuals that were less inbred than the population average. Furthermore, we found some evidence of stronger effects of inbreeding on telomere length in males than females. Thus, telomere length may reveal subtle costs of inbreeding in the wild and demonstrate a route by which inbreeding negatively impacts the physiological state of an organism already at early life-history stages.

     

Female expression of the H-2-linked sex-limited protein (Slp) due to non-H-2 genes

Female mice of 15 inbred strains in which males express the H-2-linked sex-limited protein (Slp) were tested for the production of this protein. Four inbred strains (FM, LG/J, NZB, PL/J) were found in which females produce Slp in the absence of hormonal manipulation. Crosses have been made between strains FM, NZB, or PL/J and several Slp-negative strains. Slp-typing of the F₁, F₂, and backcross progeny, as well as of a number of recombinant inbred strains, indicates that production of Slp by normal females of these strains depends upon the concurrent presence of an Slp-positive,H-t2-linked allele and of permissive alleles of one or two non-H-2 autosomal genes. Complementation studies with two of the strains (FM and PL) indicate that an identical genetic mechanism mediates expression of Slp in females of these two strains. FM-derived animals carrying the testicular feminization mutation (tfm) also express Slp, as do castrated NZB mice, indicating that Slp expression in these instances is not dependent upon testosterone as it is in other inbred strains. It is concluded from these results that genes distinct from the putative structural gene for Slp influence the sex-limitation of its expression.     

Restriction fragment length polymorphisms in satellite DNA distinguish chromosomal races of the white-footed mouse Peromyscus leucopus

We describe a polymorphism revealed by a high-copy-number tandem repeat which serves to distinguish most individuals sampled (96%) from two chromosomal races of Peromyscus leucopus. Classical morphology, allozymes, mtDNA, and rDNA have all failed to provide fixed markers which separate these two chromosomal races. Data from P. leucopus further documents the utility of DNA polymorphisms to establish the natal origin (DNA ‘zipcodes’) of populations or individuals.     

Genome organization of repetitive elements in the rodent,Peromyscus leucopus

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)_n was present in 87% of the clones, (CT)_n was present in 59% of the clones, and (AT)_n and (GC)_n each was represented in our sample by a single clone (0.05%). (TCC)_n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)_n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.     

Segregating YKU80 and TLC1 Alleles Underlying Natural Variation in Telomere Properties in Wild Yeast

In yeast, as in humans, telomere length varies among individuals and is controlled by multiple loci. In a quest to define the extent of variation in telomere length, we screened 112 wild-type Saccharomyces sensu stricto isolates. We found extensive telomere length variation in S. paradoxus isolates. This phenotype correlated with their geographic origin: European strains were observed to have extremely short telomeres (<150 bp), whereas American isolates had telomeres approximately three times as long (>400 bp). Insertions of a URA3 gene near telomeres allowed accurate analysis of individual telomere lengths and telomere position effect (TPE). Crossing the American and European strains resulted in F1 spores with a continuum of telomere lengths consistent with what would be predicted if many quantitative trait loci (QTLs) were involved in length maintenance. Variation in TPE is similarly quantitative but only weakly correlated with telomere length. Genotyping F1 segregants indicated several QTLs associated with telomere length and silencing variation. These QTLs include likely candidate genes but also map to regions where there are no known genes involved in telomeric properties. We detected transgressive segregation for both phenotypes. We validated by reciprocal hemizygosity that YKU80 and TLC1 are telomere-length QTLs in the two S. paradoxus subpopulations. Furthermore, we propose that sequence divergence within the Ku heterodimer generates negative epistasis within one of the allelic combinations (American-YKU70 and European-YKU80) resulting in very short telomeres.     

Polymorphism of esterase 11 in Mus musculus,a further esterase locus on chromosome 8

A further esterase, esterase 11, which exhibits a polymorphism detectable by electrophoresis, has been observed in the house mouse, Mus musculus. In 15 inbred strains and two outbred strains, the ES-11A phenotype has been found, composed of two bands of enzyme activity of greater anodal electrophoretic mobility than the two bands of the ES-11B phenotype found in one inbred strain, one wild stock, and 101 wild mice. In F₁ hybrids (IS/Cam×C57 BL/Gr), the phenotype shown corresponds to a mixture of the two parental phenotypes. In backcrosses, ES-11 segregates as an autosomal gene, designated Es-11, closely linked to Es-2 and Es-5 on chromosome 8.This work was supported by the Medical Research Council.     

Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans

Terminal restriction fragment analysis is the only method currently available for measuring telomere length in Caenorhabditis elegans. Its limitations include low sensitivity and interference by the presence of interstitial telomeric sequences in the C.elegans genome. Here we report the adaptation of single telomere length analysis (STELA) to measure the length of telomeric repeats on the left arm of chromosome V in C.elegans. This highly sensitive PCR-based method allows telomere length measurement from as few as a single worm. The application of STELA to eight wild-type C.elegans strains revealed considerable strain-specific differences in telomere length. Within individual strains, short outlying telomeres were observed that were clearly distinct from the bulk telomere length distributions, suggesting that processes other than end-replication losses and telomerase-mediated lengthening may generate telomere length heterogeneity in C.elegans. The utility of this method was further demonstrated by the characterization of telomere shortening in mrt-2 mutants. We conclude that STELA appears to be a valuable tool for studying telomere biology in C.elegans.     

Frequent Harderian gland adenocarcinomas in inbred white-footed mice (Peromyscus leucopus)

In 1997, three lines of inbred Peromyscus leucopus--GS109A, GS16A1, and GS16B--were acquired by the Peromyscus Genetic Stock Center. Since then, records have been kept on tumors detected by visible inspection of live animals. The inbred lines GS109A and GS16A1 presented tumors with frequencies substantially higher than that of the other inbred line or of random-bred P. leucopus stock. The average age of detection was 456 +/- 75 days (n = 24) for GS109A and 568 +/- 168 days (n = 12) for GS16A1 respectively. Surprisingly, the majority of the tumors (23 of 24 for GS109A and 8 of 12 for GS16A1) appeared to be Harderian gland lesions. During the same time period only a single tumor, a fibrosarcoma, was noted in the other inbred strain (GS16B), and one Harderian gland tumor was detected in the random bred stock. On the basis of the number of animals born to each group, tumor frequencies were approximately 22.7%, 8.3%, 0.67%, and 0.07%, for GS109A, GS16A1, GS16B, and randombred P. leucopus stock, respectively. The periocular tumors appeared to be highly malignant, with elevated mitotic indices, marked anaplasia, and metastases to regional lymph nodes and lungs. The tumors were readily transplantable to other animals of the same line. Among various other species, malignant Harderian gland tumors are relatively rare.     

Changes of telomere lengths in human intracranial tumours

The termini of human chromosomes comprise stretches of G-rich repeats that are about 5–20 kilobase (kb) in length. The size of the telomeres can be determined by hybridization with probes specific for these (ttaggg)_n sequences after digestion of chromosomal DNA with appropriate restriction enzymes and electrophoretic separation of the fragments. Here, probing with the ³²P-labelled synthetic (TTAGGG)₃ oligonucleotide revealed length changes of the telomeres occurring in intracranial tumours. Among 60 samples analysed, 41.7% showed telomere elongation, and 21.7% telomere reduction, whereas 36.7% of the tumours exhibited equal lengths compared with the patients' peripheral blood leukocytes. Most of the elongated glioma telomeres exceeded in length those of untransformed astrocytes derived from human fetal tissue.     

Upregulation of telomerase function during tissue regeneration

Telomerase plays a primary role in the maintenance of telomeres in immortal, germ, and tumor cells in humans but is lacking in most somatic cells and tissues. However, many species, including fish and inbred mice, express telomerase in most cells and tissues. Little is known about the expression of telomerase in aquatic species, although the importance of telomerase for longevity has been suggested. We compared telomerase activity and telomere lengths among a broad range of tissues from aquatic species and found telomerase at significant levels in both long- and short-lived aquatic species, suggesting constitutive telomerase expression has an alternative function. Telomere lengths in these aquatic species were comparable to those observed in normal human tissues and cell strains. Given that a host of aquatic species with short life spans have telomerase and a tremendous capacity to regenerate, we tested the hypothesis that telomerase upregulation is important for tissue regeneration. During regeneration, telomerase activity was upregulated and telomere lengths are maintained with the shortest telomeres being elongated, indicating the importance for maintaining telomere length and integrity during tissue regeneration. Thus, the expression of telomerase in aquatic animals is likely not related to longevity but to their ability to regenerate injured tissue.     

Pas1 is a common lung cancer susceptibility locus in three mouse strains

Inherited predisposition to lung cancer is a phenotypic trait shared by different mouse inbred strains that show either a high or an intermediate predisposition. Other strains are instead genetically resistant. The Pas1 locus is the major determinant of lung cancer predisposition in the A/J strain (Gariboldi et al. 1993). To define the determinants of susceptibility to lung tumorigenesis in the highly susceptible SWR/J and in the intermediately susceptible BALB/c mice, we analyzed (BALB/c × SWR/J)F₂ and (BALB/c × C3H/He)F₂ crosses by genetic linkage experiments. The present results provide unequivocal evidence that the same Pas1/+ allele that leads to lung cancer predisposition is shared by A/J, SWR/J, and BALB/c strains. The intermediate susceptibility of the BALB/c strain would result by interaction of Pas1 locus with lung cancer resistance loci. Received: 18 April 1997 / Accepted: 15 June 1997     

A genomic clone containing a telomere array maps near the centromere of mouse Chromosome 6

A lambda clone of mouse DNA containing a short array of telomere hexamers has been localized by FISH to a region close to the centromere of Chromosome (Chr) 6. Amplification of DNA with primers flanking an SSR showed that most inbred strains carry one of two alleles, although five other alleles were found among the inbred strains and 11 other alleles were found in wild-derived mice. Analysis of the DNA from four Robertsonian translocations suggests that the amplified sequence is still present in these chromosomes. The finding of two fragments associated with the Sig mutant suggests that the clone lies within a congenic region created when the mutant, obtained in a (C3H x 101)F₁, was back-crossed to C57BL/6J. This region might include all or part of the centromere. Comparison of the segregation of the amplification product with the segregation of centromeric heterochromatin in an interspecies backcross, (C57BL/6 x M. spretus)F₁ x M. spretus, (BSS) shows 1/72 recombinants with the centromeric heterochromatin, while 1/62 recombinants occurred in a BSB backcross. Analysis of other loci at the proximal end of Chr 6 gives the combined map Hc6-0.73-D6Mit86-0.73-D6Rp2-2.2-D6Mitl-2.2-Wnt2-3.0-Cpa. Data from a third cross show that Cola2 lies between D6Mit82 and D6Rp2. The portion of the telomere array, Tel-rs3, that has been sequenced contains only 13/31 repeats of the consensus sequence. A variety of sequence changes from the consensus hexamer suggests that this array has been removed for a long time from evolutionary pressures to retain the TTAGGG sequence.     

Strain‐dependent variations in visceral sensitivity: relationship to stress,anxiety and spinal glutamate transporter expression

Responses to painful stimuli differ between populations, ethnic groups, sexes and even among individuals of a family. However, data regarding visceral pain are still lacking. Thus, we investigated differences in visceral nociception across inbred and outbred mouse strains using colorectal distension. Anxiety and depression‐like behaviour were assessed using the open field and forced swim test as well as the corticosterone stress response. Possible mechanistic targets [excitatory amino acid transporter (EAAT‐1), brain‐derived neurotrophic factor (BDNF) and 5HT1A receptor] were also assessed using quantitative real‐time polymerase chain reaction. Adult, male, inbred and outbred mouse strains were used in all assays (inbred strains; CBA/J Hsd, C3H/HeNHsd, BALB/c OlaHsd, C57 BL/6JOlaHsd, DBA/2J RccHsd, CAST/EiJ, SM/J, A/J OlaHsd, 129P2/OlaHsd, FVB/NHan Hsd and outbred strains: Swiss Webster, CD‐1). mRNA expression levels of EAAT‐1, BDNF and 5HT1A receptor (HTR1A) were quantified in the lumbosacral spinal cord, amygdala and hippocampus. A significant effect of strain was found in visceral sensitivity, anxiety and depressive‐like behaviours. Strain differences were also seen in both baseline and stress‐induced corticosterone levels. CBA/J mice consistently exhibited heightened visceral sensitivity, anxiety behaviour and depression‐like behaviour which were associated with decreased spinal EAAT‐1 and hippocampal BDNF and HTR1A. Our results show the CBA/J mouse strain as a novel mouse model to unravel the complex mechanisms of brain–gut axis disorders such as irritable bowel syndrome, in particular the underlying mechanisms of visceral hypersensitivity, for which there is great need. Furthermore, this study highlights the importance of genotype and the consequences for future development of transgenic strains in pain research.     

The relative lengths of individual telomeres are defined in the zygote and strictly maintained during life

Previous studies have indicated that average telomere length is partly inherited ( Slagboom et al., 1994 ; Rufer et al., 1999 ) and that there is an inherited telomere pattern in each cell ( Graakjaer et al., 2003 ); ( Londoño‐Vallejo et al., 2001 ). In this study, we quantify the importance of the initially inherited telomere lengths within cells, in relation to other factors that influence telomere length during life. We have estimated the inheritance by measuring telomere length in monozygotic (MZ) twins using Q‐FISH with a telomere specific peptide nucleic acid (PNA)‐probe. Homologous chromosomes were identified using subtelomeric polymorphic markers. We found that identical homologous telomeres from two aged MZ twins show significantly less differences in relative telomere length than when comparing the two homologues within one individual. This result means that towards the end of life, individual telomeres retain the characteristic relative length they had at the outset of life and that any length alteration during the lifespan impacts equally on genetically identical homologues. As the result applies across independent individuals, we conclude that, at least in lymphocytes, epigenetic/environmental effects on relative telomere length are relatively minor during life.     

Genetic considerations in the effects of ethanol in mice. II. A trans-acting inducibility regulator(s) affecting alcohol dehydrogenase (ADH) activity

The inducibility of alcohol dehydrogenase (ADH) has been recognized in different systems including maize, Drosophila, and mice. Our earlier results showed strain-specific ADH responses to chronic ethanol administration relative to matched littermate controls in mice. For this study we used two strains which showed induction (BALB/c and S.W.) and two strains which showed repression (C57BL/6J and 129/ReJ) to produce three sets of F₁ hybrids and their reciprocals and one set (BALB/c×C57BL/6J) of recombinant inbred (RI) lines. The ADH properties of the resulting genotypes were again evaluated following 15% ethanol treatment in drinking water (2 weeks) in relation to their littermate matched controls in replicated trials. Our F₁ results suggest complete dominance for induction over repression at the phenotypic level, and the two repressed strains showed complementation. No significant differences were observed in the reciprocal F₁'s and all pairs of a given genotype-treatment combination yielded consistent results. The 1:1 segregation of RI lines suggests a single gene difference for ADH inducibility between BALB/c and C57BL/6J. These findings suggest the presence of a trans-acting inducibility regulator(s) for ADH which may or may not represent a single locus. Variability for such regulatory elements may provide an explanation for the commonly observed individual differences in natural populations for response to alcohol including alcohol metabolism.     

Genotype-specific environmental impact on the variance of blood values in inbred and F1 hybrid mice

Mice are important models for biomedical research because of the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Inbred mouse strains as well as F₁ hybrid mice are routinely used as genetically defined animal models; however, only a few studies investigated the variance of phenotypic parameters in inbred versus F₁ hybrid mice and the potential interference of the genetic background with different housing conditions. Thus, we analyzed the ranges of clinical chemical and hematologic parameters in C3H and C57BL/6 inbred mice and their reciprocal F₁ hybrids (B6C3F1, C3B6F1) in two different mouse facilities. Two thirds of the blood parameters examined in the same strain differed between the facilities for both the inbred strains and the F₁ hybrid lines. The relation of the values between inbred and F₁ hybrid mice was also affected by the facility. The variance of blood parameters in F₁ hybrid mice compared with their parental inbred strains was inconsistent in one facility but generally smaller in the other facility. A subsequent study of F₁ hybrid animals derived from the parental strains C3H and BALB/c, which was done in the latter housing unit, detected no general difference in the variance of blood parameters between F₁ hybrid and inbred mice. Our study clearly demonstrates the possibility of major interactions between genotype and environment regarding the variance of clinical chemical and hematologic parameters.     

Rapid,pervasive genetic differentiation of urban white‐footed mouse (Peromyscus leucopus) populations in New York City

We investigated genetic diversity and structure of urban white‐footed mouse, Peromyscus leucopus, populations in New York City (NYC) using variation at 18 microsatellite loci. White‐footed mice are ‘urban adapters’ that occur at higher population densities as habitat fragments are reduced in area but have a limited ability to disperse through urbanized areas. We hypothesized that this combination of traits has produced substantial genetic structure but minimal loss of genetic variation over the last century in NYC. Allelic diversity and heterozygosity in 14 NYC populations were high, and nearly all of our NYC study sites contained genetically distinct populations of white‐footed mice as measured by pairwise F_ST, assignment tests, and Bayesian clustering analyses performed by Structure and baps . Analysis of molecular variance revealed that genetic differences between populations separated by a few kilometres are more significant than differences between prehistorically isolated landmasses (i.e. Bronx, Queens, and Manhattan). Allele size permutation tests and lack of isolation by distance indicated that mutation and migration are less important than drift as explanations for structure in urban, fragmented P. leucopus populations. Peromyscus often exhibit little genetic structure over even regional scales, prompting us to conclude that urbanization is a particularly potent driver of genetic differentiation compared to natural fragmentation.     

Effects of ENU dosage on mouse strains

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F₁ hybrids to ENU. The results show that most F₁ hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU. Received: 11 February 2000 / Accepted: 11 February 2000