Fast skeletal muscle-specific expression of a quail troponin I gene in transgenic mice.

We have produced seven lines of transgenic mice carrying the quail gene encoding the fast skeletal muscle-specific isoform of troponin I (TnIf). The quail DNA included the entire TnIf gene, 530 base pairs of 5'-flanking DNA, and 1.5 kilobase pairs of 3'-flanking DNA. In all seven transgenic lines, normally initiated and processed quail TnIf mRNA was expressed in skeletal muscle, where it accumulated to levels comparable to that in quail muscle. Moreover, in the three lines tested, quail TnIf mRNA levels were manyfold higher in a fast skeletal muscle (gastrocnemius) than in a slow skeletal muscle (soleus). We conclude that the cellular mechanisms directing muscle fiber type-specific TnIf gene expression are mediated by cis-regulatory elements present on the introduced quail DNA fragment and that they control TnIf expression by affecting the accumulation of TnIf mRNA. These elements have been functionally conserved since the evolutionary divergence of birds and mammals, despite the major physiological and morphological differences existing between avian (tonic) and mammalian (twitch) slow muscles. In lines of transgenic mice carrying multiple tandemly repeated copies of the transgene, an aberrant quail TnIf transcript (differing from normal TnIf mRNA upstream of exon 2) also accumulated in certain tissues, particularly lung, brain, spleen, and heart tissues. However, this aberrant transcript was not detected in a transgenic line which carries only a single copy of the quail gene.

Molecular cloning of troponin I expressed in fast white muscle of a teleost fish, the Atlantic herring (Clupea harengus L.).

By cDNA cloning and 5'-RACE analysis we characterised a Clupea harengus troponin I mRNA expressed in larvae and in adult white (fast) muscle, but not in red (slow) or cardiac muscle. The mRNA encodes a TnI protein of the short chain length (176 residues) N-terminally truncated type previously observed only in tetrapod skeletal muscles. Despite its expression specificity the herring TnI does not particularly resemble the tetrapod fast skeletal muscle isoform in sequence but appears to be outside of the tetrapod TnIfast/Tnslow/TnIcardiac isoform family. Surprisingly, the actin/TnC-binding sequence resembles that of arthropod, rather than tetrapod vertebrate, troponin I's and has, besides, unique features not seen in any other troponin I's, vertebrate or invertebrate.

Human cytomegalovirus IE1 promoter/enhancer drives variable gene expression in all fiber types in transgenic mouse skeletal muscle.

BACKGROUND: Versatile transgenic manipulation of skeletal muscle requires knowledge of the expression profiles of diverse promoter/enhancer elements in the transcriptionally specialized fiber types of which muscle is composed. "Universal" viral promoters/enhancers, e.g., cytomegalovirus IE1 (CMV IE1), are of interest as reagents that may drive broad expression. However, a previous study noted a marked heterogeneity of CMV IE1-driven transgene expression among muscle fibers, raising the possibility of fiber-type-restricted expression. The purpose of the present study was to characterize CMV IE1-driven expression in terms of fiber type. RESULTS: We produced two lines of transgenic mice carrying the CMV IE1/ beta-galactosidase construct CMVLacZ, and analyzed transgene expression and fiber type by histochemical analysis of hindlimb muscle sections. In both lines CMVLacZ was expressed in all four major fiber types: type I (slow) and types IIA, IIB and IIX (fast). There was no unique pattern of fiber-type-preferential expression; fiber-type quantitative differences were observed but details varied between muscle regions and between lines. Both lines showed similar fiber-type-independent regional differences in overall expression levels, and a high level of within-fiber-type variability of expression, even among nearby fibers. The soleus muscle showed strong expression and comparatively little within-fiber-type or between-fiber-type variability. CONCLUSIONS: The CMV IE1 promoter/enhancer is not fiber-type-restricted and can be useful for driving germ-line transgene expression in all four fiber types. However, not all fibers express the gene at high levels due in part to regional differences in overall expression levels, and to a high level of within-fiber-type variability. Given the multinucleate syncitial nature of muscle fibers, it is not likely that this variability is due to variegating heterochromatinization. The soleus muscle would make a suitable subject for near-uniform experimental gene expression driven by CMV IE1 elements.

Health care reform: we need it, but do we have the national will to shape our future?

The problems in our health care system were not addressed legislatively, and they still exist despite many state and private sector attempts to control costs while increasing access. The public was seldom exposed to widespread discussion of the complex and interrelated problems in the current health care system, and consequently did not understand the need for reform, the various strategies being proposed, or the language of reform. It was easy for special interest groups to scare an untrusting public into believing that the status quo was safer than change. Despite the massive U.S. spending on health care, the population's health status does not appear to justify the investment. Prevention may be the single most effective long-term cost containment strategy available. Nurses can play a vital role in shaping health policy by engaging and educating the public, and helping to educate state and federal legislators.

Molecular evolution of the vertebrate troponin I gene family.

In the higher vertebrates troponin I (TnI) is encoded by three related genes, each of which is expressed specifically in one of the three major sarcomeric muscle cell classes, i.e. cardiomyocytes or fast or slow skeletal muscle fibers. The TnIcardiac isoform contains an "extra" block of proline-rich protein sequence near the N-terminus encoded by an exon that has no counterpart in the TnIfast and TnIslow genes. All three TnI isoforms appear to be orthologously related between birds and mammals, indicating that the TnI gene family was already established in its modern form in the early reptile common ancestor to birds and mammals. Analysis of ascidian TnI suggests that early vertebrate ancestors contained a single TnI gene and that the gene duplications that established the family occurred after the ascidian/vertebrate divergence. Evidence from organisms representing evolutionary intermediates between ascidians and reptiles is incomplete and does not yet delineate the exact order and timing of the TnI gene duplication events. However it does appear that early tetrapods already contained specialized TnI genes encoding long and short isoforms and that multiple differentially expressed TnI genes were present in the vertebrate lineage before the teleost/tetrapod divergence. Ascidians and the protostome invertebrate Drosophila produce long and short TnI isoforms (the longer isoforms containing a proline-rich block of extra sequence near the N-terminus) by an alternative RNA splicing mechanism from a single gene. It is likely that the alternative splicing mechanism is an ancestral feature, and that during vertebrate evolution this mechanism was abandoned in favor of transcriptional regulatory mechanisms directing tissue-specific expression of multiple genes separately encoding long and short TnI isoforms.

Strong evolutionary conservation of broadly expressed protein isoforms in the troponin I gene family and other vertebrate gene families.

It is well established that different protein classes undergo molecular evolution at different rates, presumably reflecting differing functional constraints. However, it is also the case that different isoforms of the "same" protein, encoded by a multigene family, may evolve at different rates. Here I report a relationship within gene families between isoform evolutionary rate and gene expression profile: Broadly expressed isoforms show stronger sequence conservation than do narrowly expressed isoforms. This observation emerged initially from cDNA cloning and sequencing studies, described here, of a vertebrate gene family encoding three differentially expressed isoforms of the muscle protein troponin I. However, the expression breadth/sequence conservation relationship applies to vertebrate gene families in general. In a broad and arbitrary survey sampling of sequence data on well-characterized vertebrate gene families, I found that in 14/15 families the most strongly conserved isoform was the most broadly expressed isoform, or one of several similarly broadly expressed isoforms. Broadly expressed isoforms are presumably subjected to greater negative selection pressure because they must function in a more diverse biochemical environment than do narrowly expressed isoforms. The expression breadth/evolutionary rate relationship has several interesting implications regarding the overall process of gene family evolution by duplication/divergence from ancestral genes.

Striated muscle-type tropomyosin in a chordate smooth muscle, ascidian body-wall muscle.

Body-wall muscle tropomyosin (Tm) of a marine chordate, the ascidian Ciona intestinalis, was studied by protein and cDNA clone analyses. Our results indicate that body-wall muscle of Ciona contains one major Tm isoform encoded by a single gene. Unexpectedly, the sequence of this Tm resembles vertebrate-striated muscle Tm isoforms, rather than those of smooth muscle or nonmuscle tissues, despite the fact that body-wall muscle is a nonsarcomeric (i.e. smooth) muscle. We also found that an apparently identical Tm isoform, derived from the same gene, is expressed at high levels in Ciona heart, a striated muscle. This is the first example of an organism in which a single Tm isoform is prominently expressed in both sarcomeric and non-sarcomeric tissues. Our results demonstrate that the characteristic features of "sarcomeric" Tm isoforms are not primarily related to sarcomeric ultrastructure per se. Instead, because ascidian body-wall muscle, unlike vertebrate smooth muscle, contains troponin, we suggest that it is the interaction with troponin that generates the selective pressure to maintain the characteristic C-terminal structure of so-called sarcomeric Tm isoforms. Our results further document the remarkable molecular similarity between the nonsarcomeric ascidian body-wall muscle and vertebrate-striated muscle. We suggest that these muscle types represent sarcomeric and nonsarcomeric variants of a fundamental class of troponin/Tm-regulated muscles, contrary to the traditional smooth/striated classification of muscle types. The possible relationship of this class of muscle to vertebrate smooth muscle is discussed.

Similar sets of terminal oligonucleotides from reovirus double-stranded RNA and viral messenger RNA synthesized in vitro.

Reovirus genomic double-stranded RNA (dsRNA) and viral messenger RNA synthesized in vitro were labeled by periodate oxidation and [3H]borohydride reduction. The 3H label was incorporated into 3'-terminal C residues and 5'-terminal N7-methylguanosine residues. Analysis of ribonuclease digests of the 3H-labeled RNA indicated that the minus strands of dsRNA contained the common 3'-terminal sequence ... PypPupGpC and that the plus strands contained heterogeneous sequence at both the 5' termini and 3'termini that corresponded to sequences at the 5' termini and 3' termini of mRNA. These results suggest that the plus strands of dsRNA and mRNA synthesized in vitro are essentially identical sets of molecules.

Codon usage in muscle genes and liver genes.

Synonymous codon usage frequencies, derived from cDNA clone sequences, were compared for several sets of vertebrate genes. Gene sets as diverse as those expressed in avian skeletal muscle and in mammalian liver showed similar patterns of synonymous codon usage. There were no significant differences suggesting tissue-specific co-adaptation of codon usage patterns and tRNA anticodon profiles. The results indicate a consensus codon usage pattern for vertebrate genes which is largely independent of taxonomic class, tissue of expression, and the cellular fate and rate of evolution of the encoded proteins. Certain elements of the consensus codon usage pattern indicate that it is the product of natural selection and not simply a mutational equilibrium among phenotypically equivalent synonyms.

5' Terminal noncoding sequence heterogeneity in reovirus mRNA.

The nucleotide sequences of the mRNAs of reovirus appear to diverge near the 5' termini. Ribonuclease T1 digestion of methylated mRNA synthesized in vitro yielded seven different 5' terminal fragments of the form m7G5'pp5' GmpCpUp(Np)nGp. Chain length analysis showed that the parameter "n" in this structural formula assumes the values 3, 4 and 5.

cDNA clone analysis of six co-regulated mRNAs encoding skeletal muscle contractile proteins.

A cDNA cloning approach was used to investigate muscle gene regulation during differentiation of cultured embryonic quail myoblasts. A cDNA clone library of cultured myofiber poly(A)+RNA was constructed and screened by colony hybridization with cDNA probes of myoblast and myofiber RNA. Twenty-eight myofiber-specific cDNA clones were identified and, by cross-hybridization analysis, these clones were found to represent, at most, 18 different myofiber-specific RNAs. Six of these RNAs were identified by sequence analysis of the cDNA clones. These six RNAs encode the contractile proteins alpha-actin, alpha-tropomyosin, myosin heavy chain, myosin light chain 2, troponin C, and troponin I. The embryonic muscle contractile protein sequences are identical with, or closely match, those of adult skeletal muscle proteins and include both fast fiber (myosin light chain 2 and troponin I) and slow fiber (troponin C) isotypes. RNA gel transfer hybridization analysis showed that the cellular abundances of these contractile protein mRNAs increase 20- to 30-fold or more during myoblast differentiation. These findings indicate that coordinate activation of contractile protein synthesis during myogenesis is controlled by mechanisms that direct the accumulation of contractile protein mRNAs rather than their translational utilization. Furthermore, with the possible exception of myosin heavy chain, the contractile protein genes expressed by cultured embryogenic muscle encode adult muscle proteins of both basal and slow fiber types, consistent with a co-activation-selective repression model of gene regulation during fiber type differentiation in developing skeletal muscle.

Nucleotide sequences at the 5' termini of reovirus mRNA's.

During in vitro synthesis of reovirus mRNA by viral cores, methyl groups from S-adenosylmethionine are incorporated only into 5'-terminal cap structures, i.e., m7GpppGmCp.... Thus, mRNA synthesized in the presence of S-adenosyl-[methyl-3H]methionine is 3H labeled specifically at the 5' terminus. This circumstance was exploited in the determination of 5'-terminal nucleotide sequences. Seven 5'-terminal fragments derived by complete RNase T1, digestion of methyl-3Hlabeled mRNA were partially degraded with RNase T2, and the products were separated by electrophoresis-homochromatography. From the patterns formed by the methyl-3H-labeled RNase T2 products, the sequences of the seven RNase T1-generated fragments were deduced. All seven fragments started with the sequence m7GpppGmCUA, after which the sequences diverged, with a tendency to be either U-rich or A-rich. Their chain lengths ranged from 7 to 10 nucleotides (excluding the m7G residue), and none of them contained an initiator AUG triplet. The sequences obtained support the hypothesis that virion-associated oligonucleotides arise through abortive transcription of the viral genome. There is no apparent 5'-terminal sequence feature distinctive of early versus late mRNA species within the small-mRNA size class.

mRNA 5'-leader trans-splicing in the chordates.

We report the discovery of mRNA 5'-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5'-leader apparently derived from a 46-nucleotide RNA that shares features with previously characterized splice donor SL RNAs. SL trans-splicing was known previously to occur in several protist and metazoan phyla, however, this is the first report of SL trans-splicing within the deuterostome division of the metazoa. SL trans-splicing is not known to occur in the vertebrates. However, because ascidians are primitive chordates related to vertebrate ancestors, our findings raise the possibility of ancestral SL trans-splicing in the vertebrate lineage.

Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice.

We analyzed, in transgenic mice, the cellular expression pattern of the quail fast skeletal muscle troponin I (TnIfast) gene and of a chimeric reporter construct in which quail TnIfast DNA sequences drive expression of E. coli beta-galactosidase (beta-gal). Both constructs were actively expressed in skeletal muscle and specifically in fast, as opposed to slow, muscle fibers. Unexpectedly, both constructs showed a marked differential expression among the adult fast fiber subtypes according to the pattern IIB > IIX > IIA. This expression pattern was consistent in multiple lines and differed from the endogenous mouse TnIfast pattern, which shows approximately equal expression in all fast fibers. These observations indicate that distinct regulatory mechanisms contribute to high-level expression of TnIfast in the various fast fiber subtypes and suggest that the outwardly simple pattern of equal expression in all fast fiber types shown by the endogenous mouse TnIfast gene is based on an intricate system of counterbalancing mechanisms. The adult expression pattern of the TnIfast/beta-gal construct emerged in a two-stage developmental process. Differential expression in fast versus slow fibers was evident in neonatal animals, although expression in fast fibers was relatively weak and homogeneous. During the first two weeks of postnatal life, expression in maturing IIB fibers was greatly increased whereas that in IIA/IIX fibers remained weak, giving rise to marked differential expression among fast fiber types. Thus at least two serially acting (pre- and post-natal) fiber-type-specific regulatory mechanisms contribute to high-level gene expression in adult fast muscle fibers. Unexpected similarities between TnIfast transgene expression and that of the myosin heavy chain gene family (which includes differentially expressed IIB-, IIX- and IIA-specific members) suggest that similar mechanisms may regulate adult fast muscle gene expression in a variety of unrelated muscle gene families.

Skeletal muscle gene transfer: regeneration-associated deregulation of fast troponin I fiber type specificity.

Direct gene transfer into skeletal muscle in vivo presents a convenient experimental approach for studies of adult muscle gene regulatory mechanisms, including fast vs. slow fiber type specificity. Previous studies have reported preferential expression of fast myosin heavy chain and slow myosin light chain and troponin I (TnIslow) gene constructs in muscles enriched in the appropriate fiber type. We now report a troponin I fast (TnIfast) direct gene transfer study. We injected into the mouse soleus muscle plasmid DNA or recombinant adenovirus carrying a TnIfast/ beta-galactosidase (beta-gal) reporter construct that had previously been shown to be expressed specifically in fast fibers in transgenic mice. Surprisingly, microscopic histochemical analysis 1 and 4 wk postinjection showed similar TnIfast/beta-gal expression in fast and slow fibers. A low but significant level of muscle fiber segmental regeneration was evident in muscles 1 wk postinjection, and TnIfast/beta-gal expression was preferentially targeted to regenerating fiber segments. This finding can explain why TnIfast constructs are deregulated with regard to fiber type specificity, whereas the myosin constructs previously studied are not. The involvement of regenerating fiber segments in transduction by plasmid DNA and recombinant adenoviruses injected into intact normal adult muscle is an unanticipated factor that should be taken into account in the planning and interpretation of direct gene transfer experiments.

Tissue-specific alternative splicing of ascidian troponin I isoforms. Redesign of a protein isoform-generating mechanism during chordate evolution.

In vertebrates, troponin I (TnI) exists as shorter and longer isoforms encoded by distinct genes expressed in skeletal and cardiac muscle, respectively. We report that the protochordate ascidian Ciona intestinalis expresses a homologous set of shorter and longer TnI isoforms in body wall muscle and heart, respectively. The heart-specific segment of the ascidian longer TnI isoform shares several sequence features with vertebrate cardiac TnI but lacks the protein kinase A phosphorylation sites implicated in sympatho-adrenal control of cardiac function. In contrast with vertebrates, the ascidian longer and shorter TnI isoforms are produced from a single gene by tissue-specific alternative RNA splicing; remarkably, the molecular mechanism of TnI isoform generation has been entirely reworked during ascidian/vertebrate evolution. Because alternative splicing is the more probable chordate ancestral condition, the long/cardiac versus short/somatic muscle pattern of TnI isoforms likely existed before the occurrence of the gene duplication events that created the vertebrate TnI gene family. Thus, gene duplication was apparently not the primary engine of isoform diversity in this aspect of TnI gene family evolution; rather, it simply provided an alternative (transcriptional) means of maintaining a previously established system of isoform diversity and tissue specificity based on alternative RNA splicing.

m7G5'ppp5'GmptcpUp at the 5' terminus of reovirus messenger RNA.

In the presence of S-adenosyl methionine the 5' terminal guanosine residue of in vitro synthesized reovirus mRNA becomes methylated at the 2'-OH position. In addition, 7-methyl guanylic acid is condensed covalently at the 5' terminus resulting in the formation of a 5' to 5' triphosphate bridge. Analysis of the 5' terminal sequence of methylated reovirus mRNA revealed that it has the structure m7G5'ppp5'GmpCpUp.