Displacement synthesis of globin complementary DNA: evidence for sequence amplification.

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.

Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions.

Physical and chemical criteria of lipoproteins containing apolipoprotein B, extracted from human aortic intima, were compared with those of plasma low density lipoproteins (LDL). Homogenates of grossly normal intima and advanced atherosclerotic lesions were subjected to differential ultracentrifugation to isolate a d = 1.006--1.063 g/ml density fraction which was extensively characterized. By electroimmunoassay, over 90% of the recovered apolipoprotein B immunological reactivity was found in isolates from both plaques and normal intima. In isolates of plaque and normal intima, particles of the same size as LDL were found, although a small population of very large structures was also present in plaque fractions. Apolipoprotein composition was similar to that of plasma LDL except for the presence of human serum albumin in aortic isolates. Fractions from aorta demonstrated greater electrophoretic mobilities than LDL. The lipid composition of isolates from normal intima was similar to that of LDL. The lipid composition of plaque fractions showed a significant decrease in the cholesteryl ester to free cholesterol ratio and in the triglyceride content in comparison to LDL and to fractions from normal intima. The fatty acid pattern of the cholesteryl ester fraction from isolates of both normal and plaque aortic homogenates demonstrated a significant decrease in the linoleate to oleate ratio as compared to LDL. Our initial studies suggest that althought aortic fractions are similar to LDL by certain criteria, some differences observed are more pronounced in fractions from lesions than from normal intima.

APO low density lipoprotein localization. Intracranial and extracranial atherosclerotic lesions from human normolipoproteinemics and hyperlipoproteinemics.

APO low density lipoprotein (apoB), the major protein in plasma low (LDL) and very low (VLDL) density lipoproteins, was localized in extracranial and intracranial arteries from normolipoproteinemics and hyperlipoproteinemics to determine if apoB extensiveness and localization varied with plasma lipoprotein profile. Specimens of carotid bifureation, internal carotid, basilar, and middle cerebral arteries from 23 subjects with normal lipoprotein levels, four with elevated LDL (type II), and 13 with elevated VLDL (type IV) values were studied with the employment of immunofluorescence techniques. Although the apoB localization pattern was identical in each group, extensiveness of positive localization was greatest in lesions from type II cases and the same in lesions from type IV and normolipoproteinemics. This suggests that sites of apoB retention are dependent on the chemical and structural changes in atherosclerotic arteries, whereas extensiveness correlates with the apoB concentration gradient between plasma and tissue.

Gene expression of an endogenous retrovirus-like element during murine development and aging.

We have measured intracisternal A-particle (IAP) RNA levels during development and aging in C57BL/6J mouse tissues to determine possible age-dependent changes in gene expression of these retrovirus-like sequences. Total RNA was isolated from tissues of embryonic and new born mice and mice ranging in age from 2 months to 32 months of age. RNA samples were either slot-blotted directly or fractionated on denaturing agarose gels and transferred to nylon membranes. Hybridization with cloned, 32P-labeled IAP sequences showed that both the mass amounts and the relative proportions of IAP transcripts varied between tissues and as a function of age. IAP gene products were higher in brain and kidney tissues than in liver and heart tissues. The relative proportion of transcripts increased in embryonic tissues until birth and following birth, was highest in neonatal or 2-month-old tissues. The adult levels of IAP-related RNAs did not change significantly from 6 to 24 months of age. However, 32-month-old tissues exhibited the lowest content of IAP transcripts, with the exception of heart tissue which did not change with age. A 5.4-kb RNA was the predominant IAP transcript in most samples, but each tissue had a characteristic size distribution of IAP-related transcripts. These results demonstrate that transcription of IAP genes continues throughout the life span of this mouse strain with tissue-specific and age-dependent regulation of expression.

Correlation in the human aorta of APO B fractions with tissue cholesterol and collagen content.

The amounts of buffer- and Triton-extracted apo B (LDL-protein), as well as the sum of these two fractions, were correlated with the total tissue cholesterol and hydroxyproline content (as a measure of collagen) in grossly normal intima, fatty streaks, and fibrous plaques of human aortas obtained at autopsy. Quantitative values of buffer- and Triton-extracted apo B were obtained by sequentially extracting homogenates of aortic intima with an aqueous buffer and one containing Triton X-100, and measuring the apo B content in each extract by an electroimmunoassay relative to plasma LDL or Triton-treated LDL. Significant positive correlations were obtained between the following: tissue cholesterol and both buffer-extracted and total-extracted apo B in grossly normal intima; tissue cholesterol and Triton-extracted apo B in microdissected fibrotic caps and cores of fibrous plaques, as well as in whole plaques. A positive correlation was also obtained between tissue cholesterol and total-extracted apo B in the necrotic core. A significant negative correlation was found between Triton-extracted apo B and collagen in whole plaques. The calculated mean percent of total tissue cholesterol in the different aortic regions that could be present as part of an intact LDL particle were: 100% in grossly normal intima, 16% in fatty streaks, and 11% in fibrous plaques. The positive correlation between Triton-extracted apo B and cholesterol in plaques suggests one or both of the following: the extracellular pool of cholesterol or some material increasing concurrently with cholesterol interacts with apo B or another part of the LDL particle; or the apo B containing lipoprotein is trapped in the hydrophobic environment of extracellular lipid. Both possibilities would render the particle less soluble in aqueous buffers. The negative correlation between Triton-extracted apo B and tissue collagen and the lack of a significant correlation between buffer-extracted apo B and collagen content suggests that collagen is probably not responsible for apo B retention in the aortic intima.

Aging affects the levels of DNA damage in postmitotic cells.

Cells are continuously exposed to DNA damaging agents that may cause mutations or lead to cell death. To counter this constant, ubiquitous attack on the genetic material, cells possess highly diverse and efficient systems to repair a variety of DNA lesions. For cells that are nondividing and are expected to remain functionally viable for many years, it is important that damage not accumulate in those genes that are essential to maintaining differentiated gene expression. If damage were to accumulate slowly in working genes, then the outcomes might appear as biological changes typically associated with senescence. Estimates on the types of DNA damage believed to arise spontaneously suggest that methylation of N7-guanine is one of the more frequently occurring events, exceeded only by single-strand breaks and possibly depurination. Previous studies have shown that the steady-state levels of m7Gua increase during aging of postmitotic mammalian tissues. To test for the possibility that repair of m7Gua might decline in senescent animals, we induced methyl adducts in young and old mice with single doses of MNU, and determined the kinetics of adduct removal. Liver, kidney and brain all exhibited some active repair of m7Gua as characterized by the rapid removal of the adduct from DNA. However, a fraction of damage was refractory to repair and was lost from DNA much more slowly. This repair-resistant fraction of damage was greater in DNA from the old tissues, but the interpretation of the data is not straightforward, because different amounts of damage were induced in young and old tissues with the same weight-normalized dose of MNU. Although old cells had higher levels of persistent adducts, initial repair rates were similar between age-matched tissues. Furthermore, experiments indicated that mRNA levels for 3-methyladenine glycosylase repair enzyme did not change with age. Our working hypothesis is that repair enzymes are present and active in senescent postmitotic tissues, but changes have occurred in old chromatin that have affected the ability of repair enzymes to efficiently process these adducts.

Identification and quantification of apolipoproteins in addition to apo[a] and apo B-100 in human lipoprotein[a].

The protein moiety of Lp[a] is widely believed to consist of one molecule of apo B-100 and one molecule of apo[a] per particle, linked by at least one disulfide bond. In this study we have re-examined the composition of Lp[a] to determine if other less abundant apolipoproteins might be present. Analysis of Lp[a] by sodium dodecyl sulfate-polyacrylamide electrophoresis under reducing conditions showed bands corresponding to < 200 kD but > 50 kD, 40 kD, 26 kD, 23 kD and 9 kD when stained with silver. Western immunoblot analysis of three preparations of Lp[a] revealed the presence of apoE and apoD. Enzyme-linked immunoassays were used to quantify apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, apoE and apo B-100 in Lp[a] and autologous LDL isolated from three healthy males. There is a significant amount of apoA-I in the Lp[a], although the levels varied widely among the different samples. ApoE concentrations were consistent in the three Lp[a] samples and were between 22 and 26% of relative apo B-100 concentrations. Relatively minor amounts of apoA-II and no apoCs were detectable in the three Lp[a] preparations. In contrast, the autologous LDL preparations contained relatively higher amounts of apoA-I, apoA-II, apoE, apoC-I, apoC-II and apoC-III. The identity of the multiple bands corresponding to < 200 kD and > 54 kD and 9 kD is not established.

Electron cryomicroscopy and digital image processing of lipoprotein(a).

Electron cryomicroscopy was used to study the structure of human lipoprotein(a) (Lp(a)), a plasma lipoprotein implicated in cardiovascular disease. An individual Lp(a) particle consists of a neutral lipid core within a shell of phospholipid, cholesterol and glycoprotein. In principle, electron cryomicroscopy images of single particles should contain structural detail attributable to the density differences among these components and the surrounding buffer. We observed such structural detail in images of frozen, hydrated Lp(a) particles. Lp(a) particles appeared to be roughly spherical in shape with an average diameter of 210 A. As is generally true for unstained samples in vitreous ice, imaged with a low electron dose, these images have low contrast with low signal-to-noise ratios. To increase the signal-to-noise ratio, we averaged classes of similar particles. We began with a set of 5813 randomly oriented Lp(a) particles and generated classes using a linear multivariate statistical method, followed by hierarchical ascendant classification. Our initial classification, based on only the first eight eigenvectors, separated particles on the basis of gross size and shape. After a rough reference-free alignment step, a second classification used the finer details in the images. This approach yielded class averages with structural detail only faintly visible in the raw, single images.

Interaction of LDL and Lp[a] with human skin fibroblasts.

We have studied the interaction of LDL and Lp[a] with fibroblasts. Our studies suggest that Lp[a] does not effectively compete with LDL for binding to the LDL receptor, and does not efficiently suppress the activity of the intracellular enzyme HMG-CoA reductase. However, Lp[a-], formed by reduction of the disulfide bond between apo[a] and apoB, behaves much like homologous LDL, whether or not apo[a] is removed from the mixture, and in spite of the fact that one or more apoB disulfides may also have been cleaved. In our studies we also noted that Lp[a] often enhanced binding of 125I-LDL by fibroblasts. Further investigation has suggested that this interaction is time-dependent. Experiments in receptor-negative fibroblasts indicate that the enhancement is not related to the presence of the LDL receptor; however, it is inhibited by the removal of calcium from the medium. The presence of sialic acid at millimolar concentrations in the medium inhibits much of the Lp[a]-enhanced binding of 125I-LDL to the cells. These studies suggest that Lp[] may in some way enhance LDL binding to cells, perhaps via interaction with cell surface glycosaminoglycans or proteoglycans or with collagen.

Bioactivation of the proximal food mutagen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) to DNA-binding species by human mammary gland enzymes.

We have investigated phase II activation of the food-derived mutagen 2-hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding of calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.

Extrachromosomal circular DNAs and genomic sequence plasticity in eukaryotic cells.

The ability of eukaryotic organisms of the same genotype to vary in developmental pattern or in phenotype according to varying environmental conditions is frequently associated with changes in extrachromosomal circular DNA (eccDNA) sequences. Although variable in size, sequence complexity, and copy number, the best characterized of these eccDNAs contain sequences homologous to chromosomal DNA which indicates that they might arise from genetic rearrangements, such as homologous recombination. The abundance of repetitive sequence families in eccDNAs is consistent with the notion that tandem repeats and dispersed repetitive elements participate in intrachromosomal recombination events. There is also evidence that a fraction of this DNA has characteristics similar to retrotransposons. It has been suggested that eccDNAs could reflect altered patterns of gene expression or an instability of chromosomal sequences during development and aging. This article reviews some of the findings and concepts regarding eccDNAs and sequence plasticity in eukaryotic genomes.

Introduction, distribution, and removal of 7-methylguanine in different liver chromatin fractions of young and old mice.

The distribution and elimination of 7-methylguanine (m7Gua) from different liver DNA chromatin fractions has been studied after treating young and old mice with N-methyl-N-nitrosourea (MNU). Guanine methylation kinetics was first studied in total liver DNA following intraperitoneal injections of 25 mg/kg and 50 mg/kg MNU does. MNU-induced DNA alkylation, as measured by m7Gua levels, was dose-dependent in liver tissues of young (9-11 month) and old mice (28-29 month). However, liver DNA in old mice incurred approximately 50% more damage than young mice for weight-normalized doses of MNU. The kinetics of adduct removal from total DNA was biphasic. A more rapid phase of m7Gua removal was observed during the first 24 h to 48 h following MNU administration; thereafter, the remaining m7Gua adducts were hydrolyzed much more slowly. Similar amounts of m7Gua were removed by 24 h at both the 25 mg/kg and 50 mg/kg MNU doses for a single age-group, but old liver tissue removed significantly more m7Gua than young liver tissue during this initial phase. Following a single injection of carcinogen (50 mg/kg), liver nuclei were isolated and chromatin was sheared by limited Micrococcal nuclease digestion. Chromatin was separated into nuclease-soluble, low-salt, high-salt and nuclear matrix fractions. All four fractions of young liver chromatin were methylated to the same degree. In contrast, there were differences in m7Gua levels between old liver chromatin fractions. DNA in the nuclease-sensitive fraction was most heavily alkylated, whereas nuclear matrix sequences were modified the least. Removal of m7Gua occurred at relatively uniform rates in all chromatin fractions regardless of age, indicating that m7Gua was not preferentially repaired in different nuclease-susceptible regions of chromatin. These results suggest that the N-methylpurine-DNA glycosylase responsible for eliminating m7Gua from the mammalian genome is not deficient in senescent liver tissue. However, there may be age-related changes in chromatin composition or structure that make some genomic sequences more accessible to alkylating agents in liver tissue of older animals.

Tissue-specific and age-related variations in repetitive sequences of mouse extrachromosomal circular DNAs.

Extrachromosomal circular (ecc) DNA was isolated from mouse brain, liver, and heart tissues at different ages. To determine the abundance of repetitive sequences in eccDNAs, preparations were probed for short-interspersed (B1 and B2), long-interspersed (L1), endogenous retroviral-like (IAP), and tandemly repeated satellite sequences (SAT) of the mouse genome. Together these sequence families comprise approximately 15% of the mouse genome. The hybridization results showed that each tissue had a characteristic pattern of repetitive sequence elements in eccDNAs, and the abundance of repetitive sequences changed as a function of age. Repetitive sequences decreased in liver and brain eccDNAs from 1 month to 8 months of age but appeared to remain stable thereafter. In contrast, repetitive sequence families in heart eccDNAs were constant from 1 month to 16 months of age but declined in 24-month-old mice. The present studies indicate that extrachromosomal sequences exhibit greater flexibility than chromosomal sequences.

Age-related studies on the removal of 7-methylguanine from DNA of mouse kidney tissue following N-methyl-N-nitrosourea treatment.

To investigate the effects of age on DNA repair of alkylation damage, C57BL/6NNia mice ranging from 9 months to 29 months of age were injected by the intraperitoneal route with single doses of N-methyl-N-nitrosourea (MNU). The rates of removal of 7-methylguanine (m7Gua) in nuclear DNA from kidney were determined at various intervals from 1 to 288 h after injection of either 25 mg or 50 mg MNU per kg body weight. Reversed phase HPLC with electrochemical detection was used to monitor adduct disappearance from DNA hydrolysates. The kinetics of m7Gua removal from DNA were at least biphasic. Evidence was obtained that there was a rapid removal of m7Gua occurring in the first 24 h after MNU administration, followed by a slow phase of removal with a t1/2 greater than 150 h. We assume that these two phases of m7Gua removal correspond to active repair of DNA by N-alkylglycosylases and to passive elimination via spontaneous hydrolysis, respectively. Young and old kidney tissues all exhibited significant repair of m7Gua (55-73% of the induced adducts were removed in the first 24 h), but a substantial fraction of m7Gua was removed slowly, indicating that there are methylated bases which were refractory to repair processes. At both doses of MNU studied, old tissues showed active repair of m7Gua that, within the limits of detection, had similar initial rates of removal as young tissues. However, old kidney did not remove this adduct with the same overall efficiency as young kidney. Therefore, the amount of m7Gua in the repair-resistant fraction was greater in the senescent tissues. The biochemical mechanisms responsible for the less efficient DNA repair in senescent kidney are not known, but we suggest that such differences are due in part to structural alterations in the chromatin.

Steady-state levels of 7-methylguanine increase in nuclear DNA of postmitotic mouse tissues during aging.

The major DNA product formed by methylating agents in vitro and in vivo is 7-methylguanine (m7Gua). In untreated rodent genomes, this damage is thought to arise as a consequence of endogenous processes. Using 2 independent HPLC systems and 2 methods of detection, we observed that low levels of m7Gua are present in nuclear DNA of normal 23-month-old postmitotic mouse tissues. We then asked whether the steady-state levels of indigenous m7Gua change as a function of age in these tissues. C57BL/6NNia male mice 11 months, 23 months, and 28 months of age were analyzed. The results showed that in nuclear DNA of brain, liver, and kidney tissues, the steady-state levels of m7Gua increased approximately 2-fold between the young and old age groups. The persistence of N-methyl-N-nitrosourea (MNU)-induced m7Gua in these tissues in treated animals was also studied. Following a 25 mg MNU/kg body weight dose, administered by the intraperitoneal route, m7Gua appeared to be at least partially persistent for a period of up to 20 days. The degree of persistence of m7Gua, however, appeared to be independent of tissue or age. Since m7Gua has intrinsic mutagenic potential and the content of m7Gua is generally a good indicator of overall alkylation damage to DNA, an age-related increase in the steady-state amounts of m7Gua may be relevant to basic mechanisms of aging and carcinogenesis.

Postlabeling analysis of indigenous aromatic DNA adducts in mouse myocardium during aging.

The possible presence of aromatic chemicals covalently linked to DNA (aromatic adducts) was investigated in heart cells during aging of the C57BL/6Nia mouse. Heart DNAs were isolated from untreated mice of different ages and analyzed by 32P-postlabeling assays. To determine low levels of adducts, assays were carried out in which aromatic adducts were first isolated by phase transfer to 1-butanol, then labeled with excess, carrier-free [gamma-32P]ATP. This analysis showed that the number and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all mouse DNA preparations and were more abundant in 32P-maps of senescent heart DNA. The results suggest that genomes of myocytes have a higher steady-state level of DNA damage in old animals which could adversely affect cell function.

Constancy of ribosomal RNA genes during aging of mouse heart cells and during serial passage of WI-38 cells.

The DNA content and ribosomal RNA gene copy number in heart of the inbred mouse strain C57BL/6 were determined at different ages. The DNA content of mouse heart remained constant, at about 150 micrograms DNA per heart, from 1 to 30 mth of age. The number of rRNA genes, as estimated by 28S rRNA . DNA hybridization, was not found to change significantly as a function of age. Likewise, the extent of rRNA hybridization to DNA from cultured human WI-38 cells at early and late passage levels was the same. These data support the notion that genomic rDNA sequences are not lost during in vivo and in vitro aging. However, the rDNA sequences are quite large and numerous small deletions or base pair substitutions would not have been detected in these studies.