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Biomedical subjects

K E Hastings

Publications and source records attributed to K E Hastings.

At least 19 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Animals↗
PubMed1998

Prior culture with concanavalin A increases intramuscular migration of transplanted myoblast.

The effect was studied of pretreatment with concanavalin A (ConA) of primary myoblast cultures on their migration when transplanted into muscles. As donors, transgenic CD1 mice in which the beta-galactosidase gene is under the control of a CMV promoter (CMVLacZ.9) were used. The myoblasts were grown with 20 microg/mL ConA during the 2 days before injecting them in the right tibialis anterior (TA) muscles of BALB/c mice and mdx mice. As a control, myoblasts from the same primary cultures were grown without ConA and injected in the left TA muscles. The host muscles were not previously irradiated or damaged by notexin injection. The recipient mice were immunosuppressed with FK506. Four days after myoblast transplantation, the area occupied by donor cells was significantly greater (more than threefold) following culture with ConA than without ConA. This result indicates that culture of myoblasts with ConA permits them to migrate farther following their transplantation in host muscles not previously damaged by notexin injection or irradiation. This suggests that pretreatment with ConA may be helpful for myoblast transplantation in humans. The mechanism of this effect still remains to be investigated.

Animals↗
PubMed1997

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.

Alternative Splicing↗
PubMed1997

Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.

Adenoviridae↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Cardiac and skeletal muscle troponin I isoforms are encoded by a dispersed gene family on mouse chromosomes 1 and 7.

We mapped the locations of the genes encoding the slow skeletal muscle, fast skeletal muscle, and cardiac isoforms of troponin I (Tnni) in the mouse genome by interspecific hybrid backcross analysis of species-specific (C57BL/6 vs Mus spretus) restriction fragment length polymorphisms (RFLPs). The slow skeletal muscle troponin I locus (Tnni1) mapped to Chromosome (Chr) 1. The fast skeletal muscle troponin I locus (Tnni2), mapped to Chr 7, approximately 70 cM from the centromere. The cardiac troponin I locus (Tnni3) also mapped to Chr 7, approximately 5-10 cM from the centromere and unlinked to the fast skeletal muscle troponin I locus. Thus, the troponin I gene family is dispersed in the mouse genome.

Animals↗
PubMed1995

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.

Health Care Reform↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Regional differences in troponin I isoform switching during rat heart development.

Expression of cardiac troponin I (TnIcardiac) and slow skeletal troponin I (TnIslow) genes was analyzed at the mRNA and protein level in the developing rat heart. TnIslow mRNA was detectable by in situ hybridization in the embryonic cardiac tube as early as the 13-somite stage (Embryonic Day 10). In contrast, TnIcardiac transcripts were first detected in the primordial atrium and ventricle of 11-day-old embryos, but were absent in the outflow tract region. TnIslow mRNA levels decreased after birth in atria and later in ventricles but persisted even in adult life in myocytes of the conduction system. TnIslow protein was detected by specific antibodies in atrial myocytes beginning from Embryonic Day 11; in contrast, ventricular myocytes were unreactive until Embryonic Day 18. Western blot analysis of 16-day-old fetal hearts confirmed the expression of TnIcardiac in atrial but not in ventricular myocardium. Slot blot analysis showed that at this stage equivalent amounts of TnIslow and TnIcardiac mRNAs are expressed in atria and ventricles. Similar differences in the expression of TnIslow and TnIcardiac mRNAs and proteins were observed in cultures of embryonic atrial and ventricular myocytes. The results suggest serial rather than simultaneous activation of TnIslow and TnIcardiac genes and they show that different regions of the developing heart differ in their patterns of TnIcardiac expression due to the operation of distinct mechanisms that separately affect the accumulation of TnIcardiac mRNA and protein.

Aging↗
PubMed1993

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.

Animals↗
PubMed1991

Structure and developmental expression of troponin I isoforms. cDNA clone analysis of avian cardiac troponin I mRNA.

We isolated avian (chicken and quail) cardiac troponin I (TnIcardiac) cDNA clones for studies of Tn-Icardiac protein structure/evolution and developmental gene regulation. Comparison of the cDNA-predicted avian TnIcardiac amino acid sequences with known TnI sequences indicated 1) that the presence of an N-terminal extension sequence carrying a dual protein kinase A phosphorylation target site and an adjacent proline-rich segment is an ancient cardiac-specific feature of TnI which has been conserved since the bird/mammal divergence, 2) that features of the near-N-terminal troponin C (TnC)-binding site sequence suggest isoform-specific adaptation of TnI and TnC, and 3) that the avian TnIcardiac internal actin/TnC-binding, actomyosin-inhibitory, domain shows significant sequence divergence from mammalian TnIcardiac sequences, including the absence of a protein kinase C target site which is a cardiac-specific feature of TnI in mammals. Use of the cDNA clones to probe TnIcardiac mRNA expression during striated muscle development showed active expression in cardiac muscle from early developmental times (day 4 in ovo), but not in embryonic or adult skeletal muscle or in embryonic skeletal muscle cell cultures. Transcriptional run-on analysis showed that the heart-specific expression of TnIcardiac mRNA in embryonic striated muscle reflects transcriptional control of TnIcardiac gene expression. In many other contractile protein gene families, genes encoding cardiac isoforms are expressed early in skeletal muscle development and are later repressed. Thus, the restriction of active TnIcardiac gene expression to the cardiac muscle cell lineage is an unusual expression pattern for cardiac contractile protein genes and indicates that diverse gene regulatory mechanisms direct the differential expression of cardiac and skeletal muscle isoforms in different muscle gene families.

Amino Acid Sequence↗