bluuebunny/crossref_metadata_2025 huggingface dataset:
Vagaries of the molecular clock
Authors: Ayala Francisco J.
Published: July 2002

The hypothesis of the molecular evolutionary clock asserts that
informational macromolecules (i.e., proteins and nucleic acids) evolve
at rates that are constant through time and for different lineages. The
clock hypothesis has been extremely powerful for determining
evolutionary events of the remote past for which the fossil and other
evidence is lacking or insufficient. I review the evolution of two
genes,
Gpdh
and
Sod
. In fruit flies, the
encoded glycerol-3-phosphate dehydrogenase (GPDH) protein evolves at a
rate of 1.1 × 10
−10
amino acid replacements per site
per year when
Drosophila
species are compared that
diverged within the last 55 million years (My), but a much faster rate
of ≈4.5 × 10
−10
replacements per site per year
when comparisons are made between mammals (≈70 My) or Dipteran
families (≈100 My), animal phyla (≈650 My), or multicellular
kingdoms (≈1100 My). The rate of superoxide dismutase (SOD) evolution
is very fast between
Drosophila
species (16.2 ×
10
−10
replacements per site per year) and remains the same
between mammals (17.2) or Dipteran families (15.9), but it becomes much
slower between animal phyla (5.3) and still slower between the three
kingdoms (3.3). If we assume a molecular clock and use the
Drosophila
rate for estimating the divergence of remote
organisms, GPDH yields estimates of 2,500 My for the divergence between
the animal phyla (occurred ≈650 My) and 3,990 My for the divergence
of the kingdoms (occurred ≈1,100 My). At the other extreme, SOD
yields divergence times of 211 My and 224 My for the animal phyla and
the kingdoms, respectively. It remains unsettled how often proteins
evolve in such erratic fashion as GPDH and SOD.
Immunity to retroviral infection: The Friend virus model
Authors: Hasenkrug Kim J. , Chesebro Bruce
Published: July 2002

Friend virus infection of adult immunocompetent mice is a well established model for studying genetic resistance to infection by an immunosuppressive retrovirus. This paper reviews both the genetics of immune resistance and the types of immune responses required for recovery from infection. Specific major histocompatibility complex (MHC) class I and II alleles are necessary for recovery, as is a non-MHC gene, Rfv-3, which controls virus-specific antibody responses. In concordance with these genetic requirements are immunological requirements for cytotoxic T lymphocyte, T helper, and antibody responses, each of which provides essential nonoverlapping functions. The complexity of responses necessary for recovery from Friend virus infection has implications for both immunotherapies and vaccines. For example, it is shown that successful passive antibody therapy is dependent on MHC type because of the requirement for T cell responses. For vaccines, successful immunization requires priming of both T cell and B cell responses.In vivodepletion experiments demonstrate different requirements for CD8+T cells depending on the vaccine used. The implications of these studies for human retroviral diseases are discussed.
Transgenic Gαq overexpression induces cardiac contractile failure in mice
Authors: D’Angelo Drew D. , Sakata Yoshihito , Lorenz John N. , Boivin Gregory P. , Walsh Richard A. , Liggett Stephen B. , Dorn Gerald W.
Published: July 2002

The critical cell signals that trigger cardiac hypertrophy and regulate the transition to heart failure are not known. To determine the role of Gαq-mediated signaling pathways in these events, transgenic mice were constructed that overexpressed wild-type Gαq in the heart using the α-myosin heavy chain promoter. Two-fold overexpression of Gαq showed no detectable effects, whereas 4-fold overexpression resulted in increased heart weight and myocyte size along with marked increases in atrial naturietic factor (≈55-fold), β-myosin heavy chain (≈8-fold), and α-skeletal actin (≈8-fold) expression, and decreased (≈3-fold) β-adrenergic receptor-stimulated adenylyl cyclase activity. All of these signals have been considered markers of hypertrophy or failure in other experimental systems or human heart failure. Echocardiography and
in vivo
cardiac hemodynamic studies indeed revealed impaired intrinsic contractility manifested as decreased fractional shortening (19 ± 2% vs. 41 ± 3%),
dP
/
dt
max, a negative force–frequency response, an altered Starling relationship, and blunted contractile responses to the β-adrenergic agonist dobutamine. At higher levels of Gαq overexpression, frank cardiac decompensation occurred in 3 of 6 animals with development of biventricular failure, pulmonary congestion, and death. The element within the pathway that appeared to be critical for these events was activation of protein kinase Cɛ. Interestingly, mitogen-activated protein kinase, which is postulated by some to be important in the hypertrophy program, was not activated. The Gαq overexpressor exhibits a biochemical and physiologic phenotype resembling both the compensated and decompensated phases of human cardiac hypertrophy and suggests a common mechanism for their pathogenesis.
Philopatry of male marine turtles inferred from mitochondrial DNA markers
Authors: FitzSimmons Nancy N. , Limpus Colin J. , Norman Janette A. , Goldizen Alan R. , Miller Jeffrey D. , Moritz Craig
Published: July 2002

Recent studies of mitochondrial DNA (mtDNA) variation among marine turtle populations are consistent with the hypothesis that females return to beaches in their natal region to nest as adults. In contrast, less is known about breeding migrations of male marine turtles and whether they too are philopatric to natal regions. Studies of geographic structuring of restriction fragment and microsatellite polymorphisms at anonymous nuclear loci in green turtle (
Chelonia mydas
) populations indicate that nuclear gene flow is higher than estimates from mtDNA analyses. Regional populations from the northern and southern Great Barrier Reef were distinct for mtDNA but indistinguishable at nuclear loci, whereas the Gulf of Carpentaria (northern Australia) population was distinct for both types of marker. To assess whether this result was due to reduced philopatry of males across the Great Barrier Reef, we determined the mtDNA haplotypes of breeding males at courtship areas for comparison with breeding females from the same three locations. We used a PCR-restriction fragment length polymorphism approach to determine control region haplotypes and designed mismatch primers for the identification of specific haplotypes. The mtDNA haplotype frequencies were not significantly different between males and females at any of the three areas and estimates of
F
st
among the regions were similar for males and females (
F
st
= 0.78 and 0.73, respectively). We conclude that breeding males, like females, are philopatric to courtship areas within their natal region. Nuclear gene flow between populations is most likely occurring through matings during migrations of both males and females through nonnatal courtship areas.
Translational coupling by modulation of feedback repression in the IF3 operon of
Escherichia coli
Authors: Chiaruttini C. , Milet M. , Springer M.
Published: July 2002

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5′ strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3′ strand are located 280 nt downstream, immediately upstream of the Shine–Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.
Carboxyfullerenes as neuroprotective agents
Authors: Dugan Laura L. , Turetsky Dorothy M. , Du Cheng , Lobner Doug , Wheeler Mark , Almli C. Robert , Shen Clifton K.-F. , Luh Tien-Yau , Choi Dennis W. , Lin Tien-Sung
Published: July 2002

Two regioisomers with
C
3
or
D
3
symmetry of water-soluble carboxylic acid C
60
derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to
N
-methyl-
d
-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the
C
3
regioisomer was more effective than the
D
3
regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 μM, the
C
3
derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The
C
3
derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Aβ
1–42
protein. Furthermore, continuous infusion of the
C
3
derivative in a transgenic mouse carrying the human mutant (
G93A
) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C
60
derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.
Neuregulin-3 (NRG3): A novel neural tissue-enriched protein that binds and activates ErbB4
Authors: Zhang Dongxiao , Sliwkowski Mark X. , Mark Melanie , Frantz Gretchen , Akita Robert , Sun Yang , Hillan Kenneth , Crowley Craig , Brush Jennifer , Godowski Paul J.
Published: July 2002

We describe the identification of Neuregulin-3 (NRG3), a novel protein that is structurally related to the neuregulins (NRG1). The NRG1/neuregulins are a diverse family of proteins that arise by alternative splicing from a single gene. These proteins play an important role in controlling the growth and differentiation of glial, epithelial, and muscle cells. The biological effects of NRG1 are mediated by receptor tyrosine kinases ErbB2, ErbB3, and ErbB4. However, genetic studies have suggested that the activity of ErbB4 may also be regulated in the central nervous system by a ligand distinct from NRG1. NRG3 is predicted to contain an extracellular domain with an epidermal growth factor (EGF) motif, a transmembrane domain, and a large cytoplasmic domain. We show that the EGF-like domain of NRG3 binds to the extracellular domain of ErbB4
in vitro
. Moreover, NRG3 binds to ErbB4 expressed on cells and stimulates tyrosine phosphorylation of this receptor. The expression of NRG3 is highly restricted to the developing and adult nervous system. These data suggest that NRG3 is a novel, neural-enriched ligand for ErbB4.
Sex-specific quantitative trait loci affecting longevity in
Drosophila melanogaster
Authors: Nuzhdin Sergey V. , Pasyukova Elena G. , Dilda Christy L. , Zeng Zhao-Bang , Mackay Trudy F. C.
Published: July 2002

Senescence, the decline in survivorship and fertility with increasing age, is a near-universal property of organisms. Senescence and limited lifespan are thought to arise because weak natural selection late in life allows the accumulation of mutations with deleterious late-age effects that are either neutral (the mutation accumulation hypothesis) or beneficial (the antagonistic pleiotropy hypothesis) early in life. Analyses of
Drosophila
spontaneous mutations, patterns of segregating variation and covariation, and lines selected for late-age fertility have implicated both classes of mutation in the evolution of aging, but neither their relative contributions nor the properties of individual loci that cause aging in nature are known. To begin to dissect the multiple genetic causes of quantitative variation in lifespan, we have conducted a genome-wide screen for quantitative trait loci (QTLs) affecting lifespan that segregate among a panel of recombinant inbred lines using a dense molecular marker map. Five autosomal QTLs were mapped by composite interval mapping and by sequential multiple marker analysis. The QTLs had large sex-specific effects on lifespan and age-specific effects on survivorship and mortality and mapped to the same regions as candidate genes with fertility, cellular aging, stress resistance and male-specific effects. Late age-of-onset QTL effects are consistent with the mutation accumulation hypothesis for the evolution of senescence, and sex-specific QTL effects suggest a novel mechanism for maintaining genetic variation for lifespan.
Operons and SL2 trans-splicing exist in nematodes outside the genus
Caenorhabditis
Authors: Evans Donald , Zorio Diego , MacMorris Margaret , Winter Carlos E. , Lea Kristi , Blumenthal Thomas
Published: July 2002

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode
Caenorhabditis elegans
contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus
Dolichorhabditis
(strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a
Dolichorhabditis
genomic region containing a gene cluster with all of the characteristics of the
C. elegans
operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the
C. elegans
and
Caenorhabditis briggsae
genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus
Caenorhabditis
from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.
The orphan nuclear receptor LXRα is positively and negatively regulated by distinct products of mevalonate metabolism
Authors: Forman Barry Marc , Ruan Benfang , Chen Jasmine , Schroepfer George J. , Evans Ronald M.
Published: July 2002

LXRα is an orphan member of the nuclear hormone receptor superfamily that displays constitutive transcriptional activity. We reasoned that this activity may result from the production of an endogenous activator that is a component of intermediary metabolism. The use of metabolic inhibitors revealed that mevalonic acid biosynthesis is required for LXRα activity. Mevalonic acid is a common metabolite used by virtually all eukaryotic cells. It serves as a precursor to a large number of important molecules including farnesyl pyrophosphate, geranylgeranyl pyrophosphate, cholesterol, and oxysterols. Inhibition of LXRα could be reversed by addition of mevalonic acid and certain oxysterols but not by other products of mevalonic acid metabolism. Surprisingly, the constitutive activity of LXRα was inhibited by geranylgeraniol, a metabolite of mevalonic acid. These findings suggest that LXRα may represent a central component of a signaling pathway that is both positively and negatively regulated by multiple products of mevalonate metabolism.
Both DNA gyrase and reverse gyrase are present in the hyperthermophilic bacterium
Thermotoga maritima
Authors: Guipaud Olivier , Marguet Evelyne , Noll Kenneth M. , de la Tour Claire Bouthier , Forterre Patrick
Published: July 2002

Like all hyperthermophiles yet tested, the bacterium
Thermotoga maritima
contains a reverse gyrase. Here we show that it contains also a DNA gyrase. The genes
top2
A and
top2B
encoding the two subunits of a DNA gyrase-like enzyme have been cloned and sequenced. The Top2A (type II DNA topoisomerase A protein) is more similar to GyrA (DNA gyrase A protein) than to ParC [topoisomerase IV (Topo IV) C protein]. The difference is especially striking at the C-terminal domain, which differentiates DNA gyrases from Topo IV. DNA gyrase activity was detected in
T. maritima
and purified to homogeneity using a novobiocin-Sepharose column. This hyperhermophilic DNA gyrase has an optimal activity around 82–86°C. In contrast to plasmids from hyperthermophilic archaea, which are from relaxed to positively supercoiled, we found that the plasmid pRQ7 from
Thermotoga
sp. RQ7 is negatively supercoiled. pRQ7 became positively supercoiled after addition of novobiocin to cell cultures, indicating that its negative supercoiling is due to the DNA gyrase of the host strain. The findings concerning DNA gyrase and negative supercoiling in Thermotogales put into question the role of reverse gyrase in hyperthermophiles.
Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance
Authors: Danielson Phillip B. , MacIntyre Ross J. , Fogleman James C.
Published: July 2002

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (
CYP28
) from two species of isoquinoline alkaloid-resistant
Drosophila
and the cosmopolitan species
Drosophila hydei
. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous
in vitro
metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification.
Molecular and functional characterization of a novel low-affinity cation transporter (LCT1) in higher plants
Authors: Schachtman Daniel P. , Kumar Raman , Schroeder Julian I. , Marsh Ellen L.
Published: July 2002

The transport of cations across membranes in higher plants plays an essential role in many physiological processes including mineral nutrition, cell expansion, and the transduction of environmental signals. In higher plants the coordinated expression of transport mechanisms is essential for specialized cellular processes and for adaptation to variable environmental conditions. To understand the molecular basis of cation transport in plant roots, a
Triticum aestivum
cDNA library was used to complement a yeast mutant deficient in potassium (K
+
) uptake. Two genes were cloned that complemented the mutant:
HKT1
and a novel cDNA described in this report encoding a cation transporter,
LCT1
(low-affinity cation transporter). Analysis of the secondary structure of LCT1 suggests that the protein contains 8–10 transmembrane helices and a hydrophilic amino terminus containing sequences enriched in Pro, Ser, Thr, and Glu (PEST). The transporter activity was assayed using radioactive isotopes in yeast cells expressing the cDNA. LCT1 mediated low-affinity uptake of the cations Rb
+
and Na
+
, and possibly allowed Ca
2+
but not Zn
2+
uptake.
LCT1
is expressed in low abundance in wheat roots and leaves. The precise functional role of this cation transporter is not known, although the competitive inhibition of cation uptake by Ca
2+
has parallels to whole plant and molecular studies that have shown the important role of Ca
2+
in reducing Na
+
uptake and ameliorating Na
+
toxicity. The structure of this higher plant ion transport protein is unique and contains PEST sequences.
Double muscling in cattle due to mutations in the myostatin gene
Authors: McPherron Alexandra C. , Lee Se-Jin
Published: July 2002

Myostatin (GDF-8) is a member of the transforming growth factor β superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal muscle mass in mice. Here we report the myostatin sequences of nine other vertebrate species and the identification of mutations in the coding sequence of bovine myostatin in two breeds of double-muscled cattle, Belgian Blue and Piedmontese, which are known to have an increase in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an 11-nucleotide deletion in the third exon which causes a frameshift that eliminates virtually all of the mature, active region of the molecule. The Piedmontese myostatin sequence contains a missense mutation in exon 3, resulting in a substitution of tyrosine for an invariant cysteine in the mature region of the protein. The similarity in phenotypes of double-muscled cattle and myostatin null mice suggests that myostatin performs the same biological function in these two species and is a potentially useful target for genetic manipulation in other farm animals.
Biological characterization of
Drosophila
Rapgap1, a GTPase activating protein for Rap1
Authors: Chen Fangli , Barkett Margaret , Ram Kavitha T. , Quintanilla Adrian , Hariharan Iswar K.
Published: July 2002

The activity of Ras family proteins is modulated
in vivo
by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the
Drosophila
Rap1 GTPase.
Drosophila
Rapgap1
encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in
Escherichia coli
, potently stimulates Rap1 GTPase activity
in vitro
. Unlike
Rap1
, which is ubiquitously expressed,
Rapgap1
expression is highly restricted.
Rapgap1
is expressed at high levels in the developing photoreceptor cells and in the optic lobe.
Rapgap1
mRNA is also localized in the pole plasm in an
oskar
-dependent manner. Although mutations that completely abolish
Rapgap1
function display no obvious phenotypic abnormalities, overexpression of
Rapgap1
induces a rough eye phenotype that is exacerbated by reducing
Rap1
gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling
in vivo
.
Disruption of the murine gene encoding phosphatidylethanolamine
N
-methyltransferase
Authors: Walkey Christopher J. , Donohue Leah Rae , Bronson Rod , Agellon Luis B. , Vance Dennis E.
Published: July 2002

All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine
N
-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene,
Pempt2
. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/−), and homozygotes (−/−) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in
Pempt2
(−/−) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from
Pempt2
(−/−) mice and a decrease in
Pempt2
(+/−) mice, compared with
Pempt2
(+/+) mice. The levels of hepatic phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [
l
-methyl-
3
H]methionine demonstrated that the
in vivo
PEMT activity was eliminated in the
Pempt2
(−/−) mice and markedly decreased in the
Pempt2
(+/−) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted
Pempt2
gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the “knockout mouse” technique to a gene for phospholipid biosynthesis.
Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication
in vitro
: Clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant
Authors: Karron Ruth A. , Buonagurio Deborah A. , Georgiu Alice F. , Whitehead Stephen S. , Adamus Jean E. , Clements-Mann Mary Lou , Harris Denos O. , Randolph Valerie B. , Udem Stephen A. , Murphy Brian R. , Sidhu Mohinderjit S.
Published: July 2002

A live, cold-passaged (
cp
) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1
cp
-52/2B5 (
cp
-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription–PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of
cp
-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the
cp
-52 virus. Nonetheless,
cp
-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication
in vivo
suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.
Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones
Authors: Wiest David L. , Bhandoola Avinash , Punt Jennifer , Kreibich Gert , McKean David , Singer Alfred
Published: July 2002

The folding and assembly of nascent proteins in the endoplasmic reticulum (ER) is assisted by molecular chaperones that are themselves retained within the ER. We now report that a number of different ER proteins, including molecular chaperones, are selectively expressed on the surface of immature thymocytes, but their surface expression is extinguished upon further differentiation. Escape from the ER is only possible for newly synthesized ER proteins before they become permanently retained. Thus, the cellular process of ER retention is incomplete in immature thymocytes and provides an explanation for surface expression of partial receptor complexes that transduce differentiative signals during thymic development.
Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic
Arabidopsis
plants brightly
Authors: Haseloff Jim , Siemering Kirby R. , Prasher Douglas C. , Hodge Sarah
Published: July 2002

The green fluorescent protein (GFP) from the jellyfish
Aequorea victoria
is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant
Arabidopsis thaliana
, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the
gfp
gene to mutate the intron and to restore proper expression in
Arabidopsis
. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed
Arabidopsis
cells and can give rise to high levels of fluorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly fluorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly fluorescent plants. These modified forms of the
gfp
gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.
Effects of a polymorphism in the human tumor necrosis factor α promoter on transcriptional activation
Authors: Wilson Anthony G. , Symons Julian A. , McDowell Tarra L. , McDevitt Hugh O. , Duff Gordon W.
Published: July 2002

Tumor necrosis factor α (TNFα) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNFα are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at −308 in the TNFα promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNFα production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at −308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNFα gene regulation and may be responsible for the association of TNF2 with high TNFα phenotype and more severe disease in infections such as malaria and leishmaniasis.
HCAR and MCAR: The human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses
Authors: Tomko Richard P. , Xu Ruliang , Philipson Lennart
Published: July 2002

The subgroup C of the adenoviruses (Ad) and the group B coxsackieviruses (CVB) are structurally unrelated viruses that are known to compete for an unidentified cell surface receptor. We now describe the isolation of cDNAs from human and mouse that encode the human CVB and Ad2 and 5 receptor (HCAR) and the mouse CVB Ad2 and 5 receptor (MCAR). Both are 46-kDa glycoproteins whose primary amino acid sequences are highly homologous. Structurally, HCAR and MCAR appear to be transmembrane proteins that contain two extracellular immunoglobulin-like domains and therefore belong to this superfamily. Transfection of either of these cDNA molecules into receptor-negative NIH 3T3 cells conferred susceptibility to CVB infection and permitted the expression of β-galactosidase from a recombinant Ad5 vector. In addition, HCAR and MCAR mRNAs could be detected on Northern blots of oligo(dT)-selected RNA from receptor-positive HeLa cells and TCMK-1 as well as several tissues of human and mouse origin that are known to be targets for Ad and CVB infections. Finally, Western blots using antibodies that inhibit virus binding to either the human or mouse CVB receptors detected 46-kDa proteins in HCAR- and MCAR-transfected cells, respectively. Taken together, these results confirm that the isolated cDNAs encode the receptors for the subgroup C Ad and CVB.
An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication
Authors: Hamy François , Felder Eduard R. , Heizmann Gerhard , Lazdins Janis , Aboul-ela Fareed , Varani Gabriele , Karn Jonathan , Klimkait Thomas
Published: July 2002

One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 × 106individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complexin vitroat nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10–30 μM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent trans-activation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, bothin vitroandin vivo.
Conserved and sexually dimorphic behavioral responses to biogenic amines in decapitated
Drosophila
Authors: Yellman Chris , Tao Heng , He Biao , Hirsh Jay
Published: July 2002

A preparation of decapitated
Drosophila melanogaster
has been used for direct application of drugs to the nerve cord. Serotonin, dopamine, and octopamine stimulate locomotion and grooming, showing distinguishable effects that often are potentiated by addition of the vertebrate monoamine oxidase-inhibitor hydrazaline. Many of the hydrazaline-induced effects are sexually dimorphic, with males showing greater responses than females. Behaviors similar to those induced by dopamine can be induced by application of the vertebrate dopamine D2-like receptor agonist quinpirole, whose effects are also sexually dimorphic. In contrast, vertebrate D2-like and D1-like dopamine antagonists result in akinesic states, and D1-like agonists selectively stimulate grooming. These data indicate that
Drosophila
nerve cord amine receptors are coupled to reflexive behaviors similar to those stimulated by brain dopamine receptors in vertebrates.
Modulation of glucose responsiveness of insulinoma β-cells by graded overexpression of glucokinase
Authors: Wang Haiyan , Iynedjian Patrick B.
Published: July 2002

Insulinoma β-cells capable of overexpressing glucokinase under the control of a doxycycline-dependent transcriptional transactivator were established from parental INS-1 cells. Glucokinase could be maximally induced to a level more than 20 times the basal level after 36 h of culture with doxycycline. Intermediate levels of induction could be achieved by varying doses of, and time of culture with, the inducer. The rate of glycolysis was measured in cells with 3-, 5-, and 8-fold increment in glucokinase activity above the noninduced level. Proportionate increases in glycolytic flux occurred in cells cultured at low physiological glucose concentration. At high glucose concentration, induction of glucokinase in excess of 2-fold above basal resulted in little additional increase in glycolysis. The consequences of graded increases of glucokinase on two physiological glucose effects were investigated. Increments in glucokinase activity were accompanied by a stepwise shift to the left of the dose–response curve for the inductive effect of glucose on the L-type pyruvate kinase mRNA. Similarly, the insulin secretory response to glucose was shifted leftward in glucokinase-induced cells. The following conclusions are drawn: (
i
) glucokinase is the major rate-limiting enzyme for glycolysis in these cells; (
ii
) downstream metabolic steps become limiting at high extracellular glucose concentration with moderate increases in glucokinase over the wild-type level; (
iii
) within limits, glucokinase activity is a determining factor for two types of glucose responses of the β-cell, the induction of specific gene expression, and insulin release.
A variable domain of delayed replication in
FRAXA
fragile X chromosomes: X inactivation-like spread of late replication
Authors: Hansen R. Scott , Canfield Theresa K. , Fjeld Alan D. , Mumm Steven , Laird Charles D. , Gartler Stanley M.
Published: July 2002

The timing of DNA replication in the Xq27 portion of the human X chromosome was studied in cells derived from normal and fragile X males to further characterize the replication delay on fragile X chromosomes. By examining a number of sequence-tagged sites (STSs) that span several megabases of Xq27, we found this portion of the normal active X chromosome to be composed of two large zones with different replication times in fibroblasts, lymphocytes, and lymphoblastoid cells. The centromere-proximal zone replicates very late in S, whereas the distal zone normally replicates somewhat earlier and contains
FMR1
, the gene responsible for fragile X syndrome when mutated. Our analysis of the region of delayed replication in fragile X cells indicates that it extends at least 400 kb 5′ of
FMR1
and appears to merge with the normal zone of very late replication in proximal Xq27. The distal border of delayed replication varies among different fragile X males, thereby defining three replicon-sized domains that can be affected in fragile X syndrome. The distal boundary of the largest region of delayed replication is located between 350 and 600 kb 3′ of
FMR1
. This example of variable spreading of late replication into multiple replicons in fragile X provides a model for the spread of inactivation associated with position-effect variegation or X chromosome inactivation.
Regulation of interleukin 4-mediated signaling by naturally occurring dominant negative and attenuated forms of human Stat6
Authors: Patel Bharvin K. R. , Pierce Jacalyn H. , LaRochelle William J.
Published: July 2002

Interleukin (IL)-4-mediated nuclear signaling by Stat6 has been implicated in lymphoid cell proliferation and the transcriptional activation of genes encoding major histocompatability complex (MHC) class II molecules and Fc receptors. To investigate IL-4-mediated transcriptional events, we cloned two naturally occurring human Stat6 isoforms, Stat6b and Stat6c, that encoded an NH2-terminal truncation or an SH2 domain deletion, respectively. Stat6 variant mRNAs were differentially expressed in many human tissues. To elucidate the biologic role of each isoform, we examined the consequences of overexpression in IL-4-responsive FDC-P2 cells. Stat6 and Stat6b (to a lesser extent) enhanced DNA synthesis, up-regulated endogenous MHC class II and Fcγ receptors, and became tyrosine phosphorylated in response to IL-4 stimulation. In contrast, Stat6c, which lacks functionally critical SH2 domain residues, unexpectedly inhibited IL-4-mediated mitogenesis and cell surface antigen expression and was not tyrosine phosphorylated. Although Stat6c only modestly diminished endogenous Stat6 tyrosine phosphorylation, it abolished endogenous Stat6 FcγRI and Iɛ DNA binding activity and FcγRI–luciferase reporter transcriptional activation. Our results indicate that the molecular mechanism of inhibition by Stat6c was due to suppression of endogenous Stat6 dimer formation. Thus, Stat6b and Stat6c are naturally occurring attenuated and dominant negative Stat6 variants, respectively, that affect IL-4-mediated biologic responses through differential transcriptional regulation.
Processing of the Ebola virus glycoprotein by the proprotein convertase furin
Authors: Volchkov Viktor E. , Feldmann Heinz , Volchkova Valentina A. , Klenk Hans-Dieter
Published: July 2002

In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse–chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGP
er
, full-length GP with oligomannosidic
N
-glycans, was detected. preGP
er
(110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP
1,2
, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP
1
, and the carboxyl-terminal 26-kDa fragment GP
2
. GP
1,2
was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.
Quantitative modeling of stochastic systems in molecular biology by using stochastic Petri nets
Authors: Goss Peter J. E. , Peccoud Jean
Published: July 2002

An integrated understanding of molecular and developmental biology must consider the large number of molecular species involved and the low concentrations of many species
in vivo
. Quantitative stochastic models of molecular interaction networks can be expressed as stochastic Petri nets (SPNs), a mathematical formalism developed in computer science. Existing software can be used to define molecular interaction networks as SPNs and solve such models for the probability distributions of molecular species. This approach allows biologists to focus on the content of models and their interpretation, rather than their implementation. The standardized format of SPNs also facilitates the replication, extension, and transfer of models between researchers. A simple chemical system is presented to demonstrate the link between stochastic models of molecular interactions and SPNs. The approach is illustrated with examples of models of genetic and biochemical phenomena where the
UltraSAN
package is used to present results from numerical analysis and the outcome of simulations.
A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export
Authors: Taura Tetsuya , Krebber Heike , Silver Pamela A.
Published: July 2002

Yeast cells mutated in
YRB2
, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin β superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.
The lepidopteran transposon vector,
piggyBac
, mediates germ-line transformation in the Mediterranean fruit fly
Authors: Handler Alfred M. , McCombs Susan D. , Fraser Malcolm J. , Saul Stephen H.
Published: July 2002

The
piggyBac
(IFP2) short inverted terminal repeat transposable element from the cabbage looper
Trichoplusia ni
was tested for gene transfer vector function as part of a bipartite vector–helper system in the Mediterranean fruit fly
Ceratitis capitata
. A
piggyBac
vector marked with the medfly white gene was tested with a normally regulated
piggyBac
transposase helper at two different concentrations in a white eye host strain. Both experiments yielded transformants at an approximate frequency of 3–5%, with a total of six lines isolated having pigmented eyes with various levels of coloration. G
1
transformant siblings from each line shared at least one common integration, with several sublines having an additional second integration. For the first transformant line isolated, two integrations were determined to be stable for 15 generations. For five of the lines, a
piggyBac
-mediated transposition was verified by sequencing the insertion site junctions isolated by inverse PCR that identified a characteristic
piggyBac
TTAA target site duplication. The efficient and stable transformation of the medfly with a lepidopteran vector represents transposon function over a relatively large evolutionary distance and suggests that the
piggyBac
system will be functional in a broad range of insects.
Characterization and solubilization of bitter-responsive receptors that couple to gustducin
Authors: Ming Ding , Ruiz-Avila Luis , Margolskee Robert F.
Published: July 2002

The tastes of many bitter and sweet compounds are thought to be transduced via guanine nucleotide binding protein (G-protein)-coupled receptors, although the biochemical nature of these receptors is poorly understood at present. Gustducin, a taste-specific G-protein closely related to the transducins, is a key component in transducing the responses to compounds that humans equate with bitter and sweet. Rod transducin, which is also expressed in taste receptor cells, can be activated by the bitter compound denatonium in the presence of bovine taste membranes. In this paper, we show that gustducin is expressed in bovine taste tissue and that both gustducin and transducin, in the presence of bovine taste membranes, can be activated specifically by several bitter compounds, including denatonium, quinine, and strychnine. We also demonstrate that the activation in response to denatonium of gustducin by presumptive bitter-responsive receptors present in taste membranes depends on an interaction with the C terminus of gustducin and requires G-protein βγ subunits to provide the receptor-interacting heterotrimer. The taste receptor–gustducin interaction can be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and transducin. Finally, as the initial step toward purifying taste receptors, we have solubilized this bitter-responsive taste receptor and maintained its biological activity.