Showing papers in "Basic life sciences in 1985"
TL;DR: It is hypothesized that loss of the equilibrium between free radical generation and antioxidant defenses results in dysdifferentiation, aging and cancer.
Abstract: There is presently no single, widely accepted, unifying theory of aging that is applicable to a wide spectrum of organisms. This fact has led many gerontologists to believe that mechanisms of aging may differ in various phylogenetic groups, albeit no compelling evidence or a priori reason has been forwarded to support this view. Given the basic similarity of molecular and cellular processes in living systems, it is indeed more probable that the contrary may be true, and that the underlying causal mechanisms of aging are essentially similar in all organisms. Conceptually, theories of aging can be assigned to two fundamentally different schools. One view regards aging as a continuation of the process of differentiation involving a programmed “shutdown” of genomic activity or a sequential activation of specific genes whose products have deleterious effects on cellular functions. Alternatively, aging is postulated to be a direct or indirect product of metabolic damage arising from the inadequacy of protective and reparative mechanisms.
124 citations
TL;DR: In this article, a conjugative mechanism was invoked to account for gene exchange because no recombinants were detected without prolonged physical contact of the parental strains (indeed, a period of mixed growth was needed) and the pattern of inheritance of groups of markers was consistent only with recombination of large segments of the parent genomes.
Abstract: Genetic exchange in Streptomyces was first revealed when prototrophic recombinants were recovered from mixed cultures of pairs of auxotrophic derivatives of several wild-type strains (1, 8, 25, 63, 65). A conjugative mechanism, rather than transformation or transduction, was invoked to account for gene exchange because no recombinants were detected without prolonged physical contact of the parental strains (indeed, a period of mixed growth was needed) and because the pattern of inheritance of groups of markers was consistent only with recombination of large segments of the parental genomes (25). Plasmids were first clearly implicated in this conjugative process in the most studied strain, Streptomyces coelicolor A3(2), when certain derivatives of the wild-type isolate were found to differ in their “fertility” properties—that is, in the frequency with which they generated chromosomal recombinants when mated with various other derivatives—and this ability was inherited “infectiously” (2, 28, 72). These experiments led to the genetic definition of two conjugative plasmids—SCP1 and SCP2—that were deduced to be present in an autonomous state in the wild-type A3(2) strain and to be lost, or in the case of SCP1 sometimes chromosomally integrated, in various of its derivatives. Plasmids responsible for “fertility” (or “chromosome mobilizing ability” [Cma]) (24) were also identified genetically in some other strains, including Streptomyces rimosus (18), Streptomyces lividans (29), Streptomyces erythreus (now called Saccharopolyspora erythrea) (15),Streptomyces venezuelae (17), and Streptomyces ambofaciens (66).
112 citations
TL;DR: The rationale for a conference on the etiology and mechanisms of aneuploidy is based upon the well-recognized association of chromosome abnormalities with human morbidity and mortality.
Abstract: The rationale for a conference on the etiology and mechanisms of aneuploidy is of course based upon the well-recognized association of chromosome abnormalities with human morbidity and mortality. Down syndrome, the best recognized phenotype, is only one of numerous adverse consequences of aneuploidy.
91 citations
TL;DR: Humans have the longest maximum lifespan potential of any mammalian species and also appear to consume more energy over this lifespan on a per-weight basis than any other species.
Abstract: Humans have the longest maximum lifespan potential of any mammalian species and also appear to consume more energy over this lifespan on a per-weight basis than any other species (Cutler, 1984a). These two unique biological characteristics of humans are the result of an unusually slow aging rate.
80 citations
TL;DR: Electron microscopy indicates that Diethylstilbestrol (DES), a synthetic estrogen, causes disruption of the mitotic spindle, centriole elongation, and unusual chromosome associations due to interkinetochore microtubules.
Abstract: The kinetochore is a structurally differentiated site on mitotic chromosomes to which spindle microtubules (MTs) are attached. In mammalian cells, the kinetochore is organized into a trilamellar plate and is morphologically distinct from the centromere. Although kinetochores and centromeres are morphologically and biochemically distinct regions, they are functionally linked and necessary for normal chromosome movement and segregation.
70 citations
TL;DR: This chapter discusses the rationale for the selection of model compounds and the final selection of a few model compounds to be used for comparative studies in diverse assays or for collaborative studies to determine interlaboratory variation of selected assays.
Abstract: There is an increasing interest in developing assay systems that are effective in detecting aneuploidy-producing agents. Because the current methodology for detecting aneuploidy is extremely varied, presently no comparisons of the validity and sensitivity of various assays can be made. This is due to a lack of sufficient data on the testing of the same compounds in multiple systems. Thus, there is an imminent need to select a few model compounds to be tested in all the available assays.
68 citations
TL;DR: These regulatory stages were reviewed by Willetts and Skurray (24), and suggestions that the F traT surface exclusion gene has its own promoter have been suggested.
Abstract: Regulation of the conjugation (tra) genes of IncF plasmids occurs by a complex, 2-stage process. Most IncF plasmids carry 2 fertility inhibition genes, finO and finP, the products of which act in concert to prevent transcription of traJ, a gene which has its own promoter (6, 22). F itself lacks finO but carries both a finP gene and the site at which FinOP regulation occurs, fisO. In turn, the traJ protein (pTraJ) is required for transcription of the major 32-kb traY→Z operon (10,22). The transfer region contains 2 further promoters, that for traM (21,23), and a weak promoter that also transcribes traI and traZ (1,23). There have also been suggestions that the F traT surface exclusion gene has its own promoter (18), as does the traT gene of R100 (16), although the polarity on surface exclusion and traD of Mu insertions into the traYZ operon argues against this (8,10). These regulatory stages were reviewed by Willetts and Skurray (24).
54 citations
TL;DR: This chapter discusses the relationship between cytogenetic abnormalities and neoplasia, especially in the context of aneuploidy in the genesis and progression of neoplastic transformation.
Abstract: This chapter discusses the relationship between cytogenetic abnormalities and neoplasia, especially in the context of aneuploidy in the genesis and progression of neoplastic transformation.
50 citations
TL;DR: A third system adds to the stability of inheritance of plasmids, theccd (control ofcelldivision) system that inhibits cell division in cells containing only 1 copy of the plasmid.
Abstract: The life cycle of bacterial plasmids involves 2 different processes, replication (rep) and partitioning (par). Both these processes have to be controlled in order to secure stable inheritance of a plasmid in a growing population of bacteria. The crucial genetic information for (control of) replication (21) and partitioning (31,41) is present on the plasmids themselves. The 2 processes seem to be independent of each other since deletion of thepar region of a plasmid does not affect the copy-number control although it results in unstable inheritance of the plasmid (41). Furthermore, thepar region of 1 plasmid can be replaced by that of another, giving totally stable inheritance and the same copy number as that of the initial replicon (42). A third system adds to the stability of inheritance of plasmids, theccd (control ofcelldivision) system that inhibits cell division in cells containing only 1 copy of the plasmid (45).
50 citations
TL;DR: The regulation of replication of the naturally occurring anti-biotic resistance plasmid R6K is concerned largely with the initiation of plasmids replication events and prevent loss of the element in an exponentially growing bacterial population.
Abstract: Bacterial plasmids vary considerably in size and copy number. A fundamental property shared by these elements, however, is their stable inheritance in a growing population of bacteria. This stable maintenance at a copy number characteristic of the element is determined by genetic regions on the plasmid that regulate the initiation of replication and subsequent partitioning of the plasmid. As in the case of bacterial and animal viruses that employ different mechanisms for their duplication in a particular host cell, plasmids have evolved different strategies to assure their maintenance at a specific copy number. In some cases both primary and secondary control circuits function to regulate the initiation of plasmid replication events and prevent loss of the element in an exponentially growing bacterial population. This paper will be concerned largely with the regulation of replication of the naturally occurring anti-biotic resistance plasmid R6K.
47 citations
TL;DR: The cause of aging is little understood at the present time, but it does appear to have a strong genetic component, reflected by the wide variation of approximately 50000 fold, in the maximal lifespan potential (MLP), seen in the various animal species.
Abstract: The cause of aging is little understood at the present time, but it does appear to have a strong genetic component. This is reflected by the wide variation of approximately 50000 fold, in the maximal lifespan potential (MLP), seen in the various animal species (Brown, 1979). Among even mammals, approximately a 100-fold variation in MLP is seen. The smokey shrew has a lifespan of only about one year, while the oldest documented human died at the age of 118, and second to him the oldest human lived to 113 (McWhirter, 1984).
TL;DR: These findings help to explain why diploid embryos with 2 male or 2 female pronuclei fail to come to term and may be connected with genetic imprinting of gametes.
Abstract: In the mouse, unbalanced gametes with major gains and/or losses of chromosomal material seem just as capable of forming a zygote as normal, fully balanced gametes. This is shown by the results of intercrossing genetically marked translocation heterozygotes, in which complementary unbalanced gametes usually fuse to form fully viable zygotes. However, there are some notable exceptions to this. Studies on a number of reciprocal translocations have shown that gametes with maternal duplication of particular chromosome regions may fail to complement those with a corresponding paternal deficiency, but produce lethal zygotes instead, whereas the reciprocal combination of a paternal duplication with a maternal deficiency produces fully viable offspring. For a particular distal region on chromosome 7 the reverse situation holds. More recent studies on genetic methods of detecting nondisjunction with Robertsonian translocations have revealed the same phenomenon. Mouse chromosomes affected include numbers 2, 6, 7, and 8. There is also defective complementation on chromosome 11 and related phenomena on chromosome 17. These findings help to explain why diploid embryos with 2 male or 2 female pronuclei fail to come to term and may be connected with genetic imprinting of gametes. It seems probable that the same phenomenon occurs in homologous regions of human chromosomes and may mean that the severity of a trisomic effect will depend sometimes on the parental source of the extra chromosome. The phenomenon also affects the efficiency of certain genetic tests for nondisjunction which depend on full complementation.
TL;DR: Primary cultures of diploid fibroblasts give rise to populations which have limited growth potential and permanent lines do not emerge from these populations, despite strong selective pressure when growth slows down and finally ceases.
Abstract: Primary cultures of diploid fibroblasts give rise to populations which have limited growth potential. In the case of human cells, their in vitro life span is usually in the range of 50–70 population doublings (Hayflick, 1965; 1977; Holliday et al., 1977). In spite of strong selective pressure when growth slows down and finally ceases, permanent lines do not emerge from these populations. Diploid rodent cultures have a much shorter in vitro life span, and permanent lines, exemplified by the mouse 3T3 strain, often take over the culture (Todaro and Green, 1963).
TL;DR: There is increasing evidence in support of the somatic mutation theory of carcinogenesis, such as the demonstration of DNA as a critical target in neoplastic transformation and the correlation between mutagenesis and carcinogenesis with most chemicals.
Abstract: There is increasing evidence in support of the somatic mutation theory of carcinogenesis (2,6,8,12–15,21,36,49,50,56,57,60,70). The recent demonstrations of the involvement of point mutations in the activation of the ras oncogenes (49,60), the associations of chromosomal rearrangements with proto-oncogenes (50,70), and the amplification of oncogenes (36,56,57) in certain tumors add to other lines of evidence, such as the demonstration of DNA as a critical target in neoplastic transformation (6,8) and the correlation between mutagenesis and carcinogenesis (2,8,12,13) with most chemicals (see Ref. 8, 41, and 51 for discussion of the arguments against the somatic mutation theory).
TL;DR: The genetic basis of the biodegradation of simple chlorinated aromatic compounds such as 4-chlorobiphenyl (pCB) or 3-, 4- or 3,5-dichloro-benzoic acids by pure cultures is reviewed, and the role of Plasmids in extending the range of chlorinated substrates that can be consumed by various bacterial genera is examined.
Abstract: Over the past several decades, man-made chlorinated aromatic compounds have been released into the environment in massive amounts in the form of herbicides, pesticides, refrigerants, lubricants or simply as industrial or hygienic household products. The presence of chlorine atoms on such molecules renders them toxic for microorganisms, insects and pests, and in some cases for human beings. The effectiveness of such compounds as insecticides or bacteriocidal agents prompted the chemical industry to manufacture varied types of the compounds and use them for enhanced agricultural productivity, various industrial processes and as health and beauty aids. The number of naturally-occurring compounds having carbon-chlorine bonds is very limited, so that microorganisms in nature have a limited capability to act upon all the complex chlorinated compounds synthesized by man. This has resulted in the persistence of these compounds and because such compounds have been widely disseminated in nature, they have created enormous problems of toxic chemical pollution, as exemplified by the episodes in the Love Canal area, the pollution in the James River or the accidental release of extremely toxic dioxins in Seveso, Italy2. Although over the years, microorganisms have been reported to slowly biodegrade various chlorinated compounds by co-oxidative metabolism3, there is still no evidence that pure cultures have acquired the ability to biodegrade highly chlorinated compounds. Reports of pure cultures capable of degrading simple mono- or dichloro compounds are now becoming available4,5. The purpose of this short article is to review the genetic basis of the biodegradation of simple chlorinated aromatic compounds such as 4-chlorobiphenyl (pCB) or 3-, 4- or 3,5-dichloro-benzoic acids by pure cultures, and examine the roleof Plasmids in extending the range of chlorinated substrates that can be consumed by various bacterial genera.
TL;DR: The use of diet to extend lifespan has so far been restricted to rodent species within the Mammals, but confirmation of the reproducibility of this effect has been reported in spite of widely differing experimental designs as mentioned in this paper.
Abstract: Subsequent to the slow historical development of the use of controlled feeding to extend maximum lifespan in rodents, interest is now focussed on exploring the potential of this animal model to understand more fully the mechanisms of aging and the biochemical etiology of chronic age-related pathologies. The use of diet to extend lifespan has so far been restricted to rodent species within the Mammals, but confirmation of the reproducibility of this effect has been reported in spite of widely differing experimental designs (Merry and Holehan, 1985a, 1985b). Controlled underfeeding such as to limit access to the normal diet so that the body weight of experimental animals is maintained at 50% of age-matched ad libitum fed rats is one of the simplest and most effective designs to delay the age at which the rate of mortality increases and results in a 36–66% extension of the maximum lifespan (Masoro et al., 1980; Merry and Holehan, 1979, 1981). Although the immediate postweaning period is not the only phase of the lifespan susceptible to the effects of underfeeding, treatments which are continued for a longer fraction of the postweaning lifespan generally have a greater effect on extending longevity. It is uncertain if the immediate postweaning period is more sensitive to the effects of underfeeding in terms of lifespan extension, but it is clear that nutrition intervention restricted to 49 days postweaning substantially reduces tumor incidence in later life (Ross and Bras, 1971).
TL;DR: Chemically induced dominant cataract mutations could be used to determine the allowable level of exposure for a single compound and the genetically characterized mutations will be an ideal source for studies in the field of developmental genetics.
Abstract: The lens is a transparent, highly refractive structure located between the pupillary portion of the iris and the vitreous A cataract is an opacity of the lens causing a reduction of visual function Ehling (1963) pointed out that morphologically comparable cataracts in mammalian species have very often the same mode of inheritance
TL;DR: Aneuploidy is the most common class of chromosome abnormality in humans and is considered by some to be the most important genetic hazard facing man.
Abstract: Aneuploidy is the most common class of chromosome abnormality in humans and is considered by some to be the most important genetic hazard facing man (2). No less than 0.3% of all newborns are aneuploid (12), most with significant physical, intellectual, and behavioral abnormalities. Furthermore, these individuals are the least affected of all aneuploid conceptions and make up only a small proportion of this class of abnormality. The vast majority of aneuploids are spontaneously aborted, with 2/3 of all chromosomally abnormal abortions being either sex chromosome monosomies (45,X) or trisomies. Together, these two classes of abnormality constitute approximately 35% of all spontaneous abortions and, assuming a minimal rate of spontaneous abortion of 15% (22), at least 5% of all clinically recognizable human pregnancies.
TL;DR: Both general- and special-purpose cloning vectors, derived from RSF1010, have proved very useful for gene manipulation in different species of soil bacteria (for review, see Ref. 4,5).
Abstract: RSF1010 is a small (8.7 kb), multicopy plasmid of the incompatibility group IncQ that confers resistances to streptomycin and sulfonamide to its host cells (1). It is very similar or identical with the 2 other representatives of the IncQ group, R300 (2) and R1162 (3). One of the most striking properties of this plasmid is its extraordinary broad host-range among gram-negative bacteria. This feature has made it attractive as a replicon for construction of vectors for gene cloning outside of Escherichia coli species. Both general- and special-purpose cloning vectors, derived from RSF1010, have proved very useful for gene manipulation in different species of soil bacteria (for review, see Ref. 4,5).
TL;DR: Studies of the dynamics of dNTP pools in 3T6 cells suggest compartmentation of dCTP pools, with perferential DNA synthesis from a pool labeled from cytidine, and experiments suggest that all dTTP formed in growing 3T 6 cells is used up for DNA synthesis.
Abstract: The replication of DNA requires a balanced supply of the four deoxyribonucleoside triphosphates (dNTPs) that is provided by reduction of the corresponding ribonucleotides through the action of the enzyme ribonucleotide reductase [24]. A single protein catalyzes the reduction of all four ribonucleotides. The evidence for this is two-fold: (i) Homogeneous proteins prepared from bacterial and mammalian sources catalyze the four reactions with about equal efficiency and (ii) single step mutations in the enzyme affect reductions of all ribonucleotides.
TL;DR: It is a quarter century since the publications of evidence for the transmissibility of drug resistance among Enterobacteria and it was that discovery that led to the great expansion of work on plasmids, their natural history, and the uses to which they can be put.
Abstract: It is a quarter century since the publications of evidence for the transmissibility of drug resistance among Enterobacteria (1,48). Although it was not the beginning of plasmid research, it was that discovery that led to the great expansion of work on plasmids, their natural history, and the uses to which they can be put. By their practical importance, resistance or “R” plasmids forced themselves on the attention of microbiologists. The biochemical mechanisms of plasmid-determined resistance were investigated for their biological interest and because of the need to counteract their effects. Because drug-resistant clones can so easily be selected from large populations of bacteria, resistance genes are used to label DNA molecules, providing a practical basis for the genetic analysis of many prokaryotic and eukaryotic systems.
TL;DR: The analysis of pT181 replication physiology has illustrated 2 important differences between directly and indirectly regulated plasmids: a) for directly regulated plasids, copy mutants specifying a normal inhibitor substance but an inactive target site exclude the wild-type or recessive mutants by directly interfering with their replication.
Abstract: PT181 is a fully sequenced Staphylococcus aureus plasmid whose size is 4,437 bp. It specifies tetracycline resistance and has a copy number of about 22 per cell in exponentially growing cultures. The functional organization of the pT181 replicon is centered around the coding sequence for a 35-kd protein, RepC, that is absolutely required for replication of the plasmid. The replication origin is contained within the repC coding sequence and the region immediately 5f to the RepC start is involved in control of the plasmid replication rate. PT181 replication is controlled at the level of RepC synthesis by a negative regulatory system that is functionally similar to that of the Co1E1 and IncFII plasmids ofEscherichia coli. The pT181 control circuit involves 2 short transcripts, RNA I and RNA II, that are transcribed from the region specifying the 5’ end of the untranslated repC mRNA leader and in the opposite direction. These are referred to as countertranscripts. The countertranscripts regulate RepC synthesis by a mechanism that probably involves interaction with the repC mRNA leader in a manner that interferes with translation. Both of the countertranscripts seem to be necessary for normal replication control; their separate roles remain unclear.
TL;DR: Maternal-age effects and their mechanisms are discussed extensively in sections that follow, including an association of advanced maternal age with the trisomy 21 phenotype, Down syndrome.
Abstract: An association of advanced maternal age with the trisomy 21 phenotype, Down syndrome, has been known for many years and has been found in all ethnic, racial, and socioeconomic groups investigated. (For references see Ret. 25 and 41.) More recently, an association of advanced maternal age with other chromosome abnormalities, both viable and nonviable, has been reported. Maternal-age effects and their mechanisms are discussed extensively in sections that follow.
TL;DR: Before the development of techniques to karyotype the spontaneously aborted conceptus, studies of human aneuploidy were based on livebirths and focused almost entirely on the most common anomalies among births: abnormalities of the sex chromosomes and trisomy 21.
Abstract: Before the development of techniques to karyotype the spontaneously aborted conceptus, studies of human aneuploidy were based on livebirths and focused almost entirely on the most common anomalies among births: abnormalities of the sex chromosomes and trisomy 21. Various studies repeatedly showed an increase in the prevalence of trisomy 21 births with maternal age, the rapid increase beginning in the mid-30s. Trisomy 13 and 18 births showed similar associations with maternal age (see Ref. 21). No other parental characteristic, including paternal age, and no environmental exposure has shown a consistent association with trisomy 21 among births.
TL;DR: The discussion of chromosome movement is begun with a description of the metaphase spindle, a dynamic and transient structure that forms to segregate the duplicate chromosomes and disappears at the completion of its task.
Abstract: The mitotic spindle is a dynamic and transient structure; it forms to segregate the duplicate chromosomes and disappears at the completion of its task. This dynamism is complex because spindle formation includes both the polymerization of fibrous components, such as microtubules, and the organization of these components into the familiar metaphase structure. The actions of the spindle fibers on chromosomes may conveniently be divided into 2 parts: an organizational phase that precedes metaphase and a segregational phase thereafter. To discuss either of these dynamic stages with clarity, it is important to have a good view of metaphase in mind. We therefore begin our discussion of chromosome movement with a description of the metaphase spindle.
TL;DR: Data suggest that Ap7* is the mature F-pilin subunit and is assembled from an inner membrane pool, which appears to require traG activity, but may also be dependent upon additional tra activities.
Abstract: Membrane fractions containing [35-S]methionine labeled proteins synthesized by Flac and Flac tra mutant strains or by λtra transducing phages expressed in such strains have been analyzed in order to investigate the pathway for synthesis of the F-pilin subunit and the gene products involved in synthesis of F-pili. Our data indicate that the synthesis of a mature F-pilin subunit requires the expression of at least 2 tra operon genes in addition to the structural gene for F-pilin, traA. In the absence of these activities, traA expression results primarily in the synthesis of a polypeptide, Apl4, with an apparent molecular weight of approximately 14,000. We assume this polypeptide corresponds to the direct product of the traA gene. In the presence of traQ activity, the major detectable product of traA is a polypeptide, Ap7(Q), which migrates with an apparent molecular weight of 7,000, suggesting that traQ product may process or assist in the processing of Apl4. Polypeptide Ap7(Q) is not, however, mature F-pilin, since it reacts poorly with anti-F-pilus-serum. Synthesis of a polypeptide which appears to be anti-genically equivalent to F-pilin and which we assume requires a modification of the F-pilin N-terminus, is detected as synthesis of a polypeptide, Ap7*. This protein migrates slightly more slowly than Ap7(Q) on our polyacrylamide gels. Polypeptide Ap7*, can be effici¬ently precipitated with F-pilus antiserum, and can be detected in both inner membrane and outer membrane fractions under conditions where assembly of F-pili can occur. These data suggest that Ap7* is the mature F-pilin subunit and is assembled from an inner membrane pool. Synthesis of Ap7* appears to require traG activity, but may also be dependent upon additional tra activities.
TL;DR: Abiotrophies share several characteristics in addition to the primary degeneration of excitable tissue which occurs in the absence of histopathologic evidence of the etiology, and may be variable in their clinical and pathological features and often overlap with one another.
Abstract: Gowers (1902) introduced the term ‘abiotrophy’ to signify the premature death of neurons and skeletal muscle in primary neuronal degenerations and in muscular dystrophies respectively. Collins (1919) classified retinitis pigmentosa, with its premature degeneration of photoreceptors, as an abiotrophy. Abiotrophies share several characteristics in addition to the primary degeneration of excitable tissue which occurs in the absence of histopathologic evidence of the etiology (Gowers, 1902; Collins, 1919; Blackwood and Corsellis, 1976; Richardson and Adams, 1977). The abiotrophic degenerations: 1) become evident after the excitable tissue has attained a normal, mature development; 2) are relentlessly progressive; 3) selectively affect certain excitable tissues but not others; 4) have either a clear hereditary or a sporadic basis; and 5) may be variable in their clinical and pathological features and often overlap with one another. Examples of abiotrophies include xeroderma pigmentosum (XP), ataxia telangiectasia, Cockayne syndrome, Alzheimer disease, Parkinson disease, Huntington disease, Friedreich ataxia, Duchenne muscular dystrophy, and retinitis pigmentosa.
TL;DR: Thymidylate synthase-negative mutants of cultured mouse FM3A cells were immediately committed to cell death upon thymidine deprivation and loss of a stably integrated human gene in mouse cells, possibly by DNA rearrangements, under the conditions where no point mutations were induced.
Abstract: Thymidylate synthase-negative mutants of cultured mouse FM3A cells were immediately committed to cell death upon thymidine deprivation especially when the cells were synchronized in the S-phase. Thymidine deprivation induced single strand breaks in parental DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with short fragments. Increase in the short DNA fragments paralleled that of thymineless death. Thymidine deprivation also accumulated double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-strand DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above short DNA fragments. Therefore, the double strand breaks seemed to occur in some defined portions of the genome and in some specific manners in contrast to those induced by X-ray, which occurred rather randomly. Cycloheximide blocked thymineless death and accumulation of the double stranded DNA fragments in parallel. The double strand breaks induced by thymidine starvation were not repaired, but instead advanced on subsequent incubation of the cells in growth medium cotaining thymidine. Cytogenetically, thymidine deprivation induced chromosome aberrations such as chromatid breaks, chromatid interchanges, and chromosome fragmentation. Also, 5-bromodeoxyridine deprivation induced sister chromatid exchange. Thymidylate stress also induced loss of a stably integrated human gene in mouse cells, possibly by DNA rearrangements, under the conditions where no point mutations were induced.
TL;DR: The above results provide strong support for the concept that respiration rate is a critical determinant of the lifespan in this organism.
Abstract: Recent work from our laboratory has provided evidence which suggests that mitochondrial disorganization may be an important aspect of age related changes of fixed postmitotic cells such as those found in the adult insect Drosophila melanogaster (Miguel et al., 1979; Fleming et al., 1982). For example, increased mitochondrial respiration in insects exposed to moderately high temperature results in both higher rates of lipopigment accumulation and life shortening (Miquel et al., 1976). Moreover, adult Drosophila showing lower respiration rates because of exposure to the mitochondrial DNA-intercalating dye ethidium bromide in their diet during development live longer than controls raised on normal medium (Fleming et al., 1981). Also, there is an inverse correlation between oxygen consumption and life span in flies treated with high levels of dietary antioxidants (Miquel et al., 1982). More recently, we have confirmed this inverse relationship for several strains of Drosophila raised under identical conditions (Miquel et al., 1985). Collectively, the above results which are shown in Fig. 1 provide strong support for the concept that respiration rate is a critical determinant of the lifespan in this organism.
TL;DR: The genetic and hormonal strategies aimed at the understanding of in vitro cellular senescence have primarily utilized cell fusion techniques and viral probes, and cell fusion studies are most consistent with a late G1 block in senescent cells.
Abstract: The characteristics of normal diploid cell proliferation are widely used to describe in vitro models of cellular aging. In fact, it is the predictable loss in proliferative capacity that was first used to argue that normal human fibroblast cultures could serve as a model system for the study of cellular aging. Although there are many other physiological changes that occur during the lifespan of cells in culture, the regulation of cell proliferation remains a primary target of research in this area of cell biology. There have been two general approaches taken in studying the regulation and eventual loss of proliferative capacity in normal human cells in culture. They can be broadly characterized as genetic and hormonal approaches.