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Biomedical subjects

A P Butler

Publications and source records attributed to A P Butler.

36 records · Page 2Linked to original sources
PubMed1994

Signal transduction pathway(s) involved in phorbol ester and autocrine induction of interleukin-1 alpha mRNA in murine keratinocytes.

We investigated the signal transduction pathways leading to the 12-O-tetradecanoylphorbol-13-acetate (TPA)- and interleukin-1 alpha (IL-1)-induced IL-1 alpha mRNA in mouse keratinocytes. Induction of IL-1 alpha mRNA by TPA or IL-1 alpha was followed by increases in cell-associated IL-1 alpha protein measured by enzyme-linked immunosorbent assay. Although protein kinase C (PKC) was involved in TPA-induced IL-1 alpha mRNA, down-regulation of PKC did not block the induction of this gene by TPA. The autocrine induction of IL-1 alpha was not mediated through PKC or cAMP. IL-1 alpha did activate mitogen-activated protein kinase. Genistein, a tyrosine kinase inhibitor, blocked both IL-1 alpha-induced mitogen-activated protein kinase activation as well as IL-1 alpha mRNA expression. Genistein, at an unsaturating dose, plus a serine/threonine kinase inhibitor, H7, completely blocked the autocrine induction of IL-1 alpha suggesting that expression of this gene is regulated by tyrosine kinase(s) in combination or independently with serine/threonine kinase(s). In addition, both TPA and IL-1 alpha caused increases not only in the phosphorylation of c-Jun and c-Fos protein but also in the transactivating activity of AP-1 nuclear transcription factor. Neither TPA nor IL-1 alpha induced NF-kappa B binding activity, as assessed by electrophoretic mobility shift analysis. This study suggests that the activation of AP-1 may be a common event through which TPA and IL-1 alpha induce IL-1 alpha mRNA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Modulation of interleukin-1 alpha mRNA expression in mouse epidermis by tumor promoters and antagonists.

Increased expression of interleukin-1 (IL-1) in skin elicits a variety of responses, including inflammation and epidermal hyperplasia, which are also characteristic events elicited by tumor promoters. The goal of this study was to investigate whether various classes of tumor promoters increase expression of IL-1 alpha and whether phorbol ester-induced IL-1 alpha expression can be blocked by antitumor promoters. Northern analysis of mRNA isolated from the dorsal skins of SENCAR mice treated with 1 microgram of 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) showed that a single application of TPA produced a significant increase in IL-1 alpha mRNA at 6 h that decreased by 24 h after treatment. Two treatments of TPA at 48-h intervals induced, at 6 h, twice as much IL-1 alpha mRNA as one treatment. Of the other promoters tested, anthralin (22.6 micrograms), mezerein (2 micrograms), calcium ionophore A23187 (120 micrograms), and benzoyl peroxide (20 mg) induced IL-1 alpha mRNA with different kinetics and to different extents. On the other hand, the non-tumor promoting phorbol ester analogue 4 alpha-12-O-tetradecanoylphorbol-13-acetate had little effect on the expression of IL-1 alpha mRNA. The effects of various antitumor promoters on TPA-induced IL-1 alpha mRNA expression were also assessed. Fluocinolone acetonide, mepacrine, and 5,8,11,14-eicosatetraynoic acid were the most effective inhibitors, and each produced about 80% inhibition. Other antitumor promoters such as retinoic acid, N-tosyl-L-phenylalanine chloromethyl ketone, and butylated hydroxytoluene inhibited approximately 35%, 65%, and 50% of TPA-induced IL-1 alpha mRNA expression, respectively. Therefore, this study suggests a possible role of IL-1 alpha in the promotion stage of skin carcinogenesis.

Animals↗
PubMed1993

Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures.

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12-O-tetradecanoylphorbol-13-acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c-jun and c-fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD-1 mice and from discarded adult human skin were grown in primary culture in a high-calcium serum-free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKC alpha, PKC beta, and PKC delta; no PKC gamma, PKC epsilon, or PKC zeta were detected. In fresh MEKs, PKC alpha, PKC beta, PKC delta, and PKC zeta were observed, but not PKC gamma or PKC epsilon. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKC gamma was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA-induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c-jun and c-fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c-jun, c-fos, and ODC mRNA in HEKs. However, c-jun and c-fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.

Adult↗
PubMed1991

Regulation of ornithine decarboxylase mRNA by phorbol esters and insulin in normal and C-kinase-deficient rat hepatoma cells.

Tumor-promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4-6-fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down-regulation or inhibition of protein kinase C (PKC), consistent with a PKC-mediated mechanism. In contrast, PKC down-regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC-depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.

Animals↗
PubMed1991

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Correlation of phorbol ester promotion in the resistant C57BL/6J mouse with sustained hyperplasia but not ornithine decarboxylase or protein kinase C.

The activation of protein kinase C, induction of ornithine decarboxylase (ODC), and hyperplasia have been suggested to be linked, sequential processes resulting from phorbol ester application to mouse skin. However, evidence is presented indicating that these events are not necessarily linked or dependent on one another and that significant differences exist in these responses between phorbol ester promotion sensitive (SSIN) and resistant (C57BL/6J) mice. The epidermis from SSIN mice treated with a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) displayed a large induction of ODC and a subsequent extensive hyperplasia. A second TPA treatment at 24 or 48 h after the first did not result in ODC induction (refractory state), and protein kinase C was shown to be down-regulated at these times. By 72 h, however, a responsive state had returned even through protein kinase C remained down-regulated. The epidermis of C57BL/6J responds to a single application of TPA with a level of ODC induction similar to that of the SSIN mice. Protein kinase C was down-regulated by approximately 75% after 24 h and was virtually completely down-regulated at 48 and 72 h (95-97%). In contrast to the above findings for the sensitive mice, however, little, if any, hyperplasia was produced. In addition, while a second TPA treatment at 24 h did not result in ODC induction (refractory state), hyperplasia did occur within 24 to 48 h. When the second TPA application was given 48 h after the first, at a time when protein kinase C was down-regulated, an overinduction of ODC occurred, as well as subsequent hyperplasia. Furthermore, a significant number of papillomas resulted when these increased treatment frequencies, i.e., once a day or every other day, were used to promote dimethylbenz(a)anthracene-initiated C57BL/6J mice. It is concluded that, while hyperplasia remains an apparent requirement for tumor promotion, the ODC induction following an initial TPA treatment is insufficient for or not causally related to this hyperplasia. In addition, subsequent ODC induction, at least in the C57BL/6J mouse, is probably not mediated by protein kinase C.

Animals↗
PubMed1987

Transient induction of ornithine decarboxylase mRNA in rat hepatoma cells treated with 12-O-tetradecanoylphorbol-13-acetate.

The contribution of changes in mRNA levels to the induction of ornithine decarboxylase (ODC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat H35 hepatoma cells was analyzed by Northern blot and quantitative dot blot hybridization. ODC mRNA accumulated rapidly in TPA-treated cultures. The increase in message was transient, reaching a peak within about 3 h, then declining to control levels after 18 h. Maximal accumulation of ODC-specific mRNA varied from 3- to 8-fold above control. The TPA dose-response for ODC message accumulation was half-maximal at approximately 0.18 microM TPA. The increase was completely blocked by actinomycin D, suggesting that TPA stimulates the transcription of ODC genes. Inhibition of protein synthesis by cycloheximide (10 micrograms/ml) led to a superinduction of ODC mRNA in the presence of TPA, which suggested that a short-lived protein may be responsible for negative control of ODC expression.

Animals↗
PubMed1986

Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2.

The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector.

Animals↗
PubMed1986

Phosphorylation of histones is stimulated by phorbol esters in quiescent Reuber H35 hepatoma cells.

Histones isolated from Reuber H35 rat hepatoma cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for possible alterations in phosphorylation. Incorporation of 32P orthophosphate into individual acid-extracted histones was monitored by autoradiography and scintillation counting of polyacrylamide gels or by reverse-phase high performance liquid chromatography. Treatment of quiescent H35 cells (arrested by serum starvation) with submicromolar doses of TPA resulted in a rapid and specific increase in phosphorylation of histones H2B and H1(0). Smaller increases in phosphorylation were observed for H4. No significant change in phosphorylation of the major H1 histones or H2A were observed after 1 h of treatment. The phosphorylation was TPA dose-dependent, with a maximum increase of approximately 14-fold for H2B, 11-fold for H1(0), and 2-fold for H4 achieved at 0.8 M TPA. The nonpromoting parent compound phorbol did not induce any of these changes. Furthermore, the mitogenic hormone insulin did not cause a similar pattern of histone phosphorylation, suggesting that the effect observed was not due to a general mitogenic response in the H35 hepatoma cells. Addition of 8-Br-cAMP also failed to reproduce the effect of TPA on histone phosphorylation, suggesting that cAMP-dependent protein kinases are not likely to be involved in mediating this response to TPA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies.

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.

Animals↗
PubMed1984

Physical structure of gene-sized chromatin from the protozoan Oxytricha.

Oxytricha nova is a hypotrichous ciliate containing a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Two-dimensional gel electrophoresis shows that macronuclei contain a normal complement of inner histones. However, despite extensive efforts, no classical H1-like protein has been detected. Micrococcal nuclease digestion indicates a nucleosome repeat length of approximately 220 bp for macronuclear chromatin. Thermal denaturation profiles of macronuclear chromatin in 0.2 mM EDTA display four transitions at about 46, 57, 64, and 79 degrees C. The lowest of these shifts to higher temperature as the ionic strength is raised to 3-5 mM Na phosphate. These results are consistent with the absence of H1 and a nucleosome repeat of 220-230 bp. Circular dichroism (CD) results agree with these findings. By contrast, micronuclear chromatin displays a much smaller premelt and a more suppressed DNA CD signal at 285 nm, consistent with a micronuclear chromatin repeat of 165-185 bp as determined by micrococcal nuclease digestion.

Animals↗
PubMed1983

Cellular and biochemical changes during multistage skin tumor promotion.

Skin tumor promotion in mice which may involve a free radical mechanism can be operationally and mechanistically further divided into at least two stages. The first stage, which is partially irreversible for 4 to 6 weeks, can be accomplished by a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or by nonpromting agents such as 4-O-methyl-TPA, calcium ionophore A23187, and hydrogen peroxide, as well as by wounding. These agents plus wounding increase the number of dark basal keratinocytes, which suggest that these cells are important in the first stage of promotion. Recent data also suggest that the dark cells may be the critical target of skin tumor initiators. Prostaglandin E2 was found to specifically enhance stage I and increase the number of dark cells induced by TPA, whereas the protease inhibitor, tosyl phenylalanine chloromethylketone, specifically inhibited stage I of promotion and counteracted the TPA-induced dark cells. We have recently found that TPA and other first stage promoters can decrease the number of epidermal glucocorticoid receptors. Fluocinolone acetamide (FA) was found to inhibit both stages but was more effective in counteracting stage I of promotion. FA also prevents the TPA-induced dark cells and the decrease in glucocorticoid receptors caused by TPA. The second stage of promotion is initially reversible but later becomes irreversible. The weak promoting agent mezerein and the nonpromoting agent 12-deoxyphorbol-13-2,4,6-decatrienoate are effective stage II promoters. Polyamines, gene amplification and epidermal cell proliferation appear to be important events in stage II of promotion. Putrescine was found to specifically enhance stage II, whereas retinoic acid, difluoromethylornithine, and butylated hydroxyanisole specifically inhibited stage II of promotion and the mezerein-induced polyamine levels but not the mezerein-induced hyperplasia. The inhibition of stage II of promotion by antioxidants gives further support for the role of free radicals in tumor promotion. Mezerein was found to be much more effective in amplifying methotrexate resistance than TPA. Although, mezerein can not significantly decrease the number of glucocorticoid receptors or increase the number of dark cells after repetitive treatment, mezerein can maintain the TPA effect in a two-stage promotion protocol.

Animals↗
PubMed1982

pH effects on the structure of the inner histones.

The structure of the inner histone complex extracted from chicken erythrocyte chromatin with 2 M NaCl has been studied as a function of pH. At pH 6, the complex dissociates to (H3-H4)2 tetramer and H2A.H2B dimer, with little change in alpha-helix content (as monitored by circular dichroism at 222 mm). Although the circular dichroism of tyrosyl side chains is also largely unchanged by the dissociation, measurements of intrinsic fluorescence do suggest a change in the environment of one or more tyrosines as a result of dissociation. Below pH 4, the histones become partially unfolded, lose specific secondary and tertiary structure, and undergo nonspecific aggregation. Both the pH 6 and 4 transitions, which are largely reversible, parallel pH-induced structural changes of nucleosomes (Zama, M., Olins, D.E., Prescott, B. and Thomas, G.J. (1978) Nucleic Acids Res. 5, 3881-3897). The results are consistent with the presence of tyrosine residues at the histone subunit-subunit contacts and suggest that histone conformation within the globular regions is largely independent of histone-DNA interactions.

Animals↗
PubMed1979

Salt-dependent interconversion of inner histone oligomers.

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.

Animals↗
PubMed1977

Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: measurement of DNA-bound Escherichia coli lac repressor in vivo.

Binding of genome regulatory proteins to nonspecific DNA sites may play an important role in controlling the thermodynamics and kinetics of the interactions of these proteins with their specific target DNA sequences. An estimate of the fraction of Escherichia coli lac repressor molecules bound in vivo to the operator region and to nonoperator sites on the E. coli chromosome is derived by measurement of the distribution of repressor between a minicell-producing E. coli strain (P678-54) and the DNA-free minicells derived therefrom. Assuming the minicell cytoplasm to be representative of that of the parent E. coli cells, we find that less than 10% of the repressor tetramers of the average cell are free in solution; the remainder are presumed to be bound to the bacterial chromosome. The minimum in vivo value of the association constant for repressor to bulk nonoperator DNA (K(RD)) calculated from these results is about 10(3) M(-1), and analysis of the sources of error in the minicell experiment suggests that the actual in vivo value of K(RD) could be substantially greater. The value of K(RD), coupled with in vitro data on the ionic strength dependence of this parameter, can be used to estimate that the effective intracellular cation activity of E. coli is no greater than about 0.24 M (and probably no less than 0.17 M) in terms of sodium ion equivalents. The minicell distribution experiments also confirm that the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA (K(RID)) is [unk] K(RD)in vivo. These results are used to calculate minimum in vivo values of K(RO) and K(RIO) (association constants for repressor and for inducer-repressor complex binding to operator) of about 10(12) M(-1) and about 10(9) M(-1), respectively. The results fit a quantitative model for operon regulation in which nonspecific DNA-repressor complexes play a key role in determining basal and constitutive levels of gene expression [von Hippel, P. H., Revzin, A., Gross, C. A. & Wang, A. C. (1974) Proc. Natl. Acad. Sci. USA 71, 4808-4812].

Bacterial Proteins↗
PubMed1977

Binding of E.coli lac repressor to non-operator DNA.

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] </= 0.002 M. Competition melting experiments are used to show that at 0.02 to 0.002 M Na(+) the affinity of lac repressor for various natural DNAs and synthetic double-stranded polynucleotides (including poly[d(m(6)A-T)], which carries a methyl group in the large groove) are approximately independent of base composition, except that the affinity of repressor for poly[d(A-T)] is approximately 2- to 3-fold greater than for the other DNAs tested. The affinity for single-stranded polynucleotides is atleast 50-fold less than for the doublehelical forms.

Bacterial Proteins↗
PubMedDate not supplied

The DNA sequence and comparative analysis of human chromosome 20.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

Animals↗