bluuebunny/crossref_metadata_2025 huggingface dataset:
TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA
Authors: Spangler Lori , Wang Xiaoxue , Conaway Joan W. , Conaway Ronald C. , Dvir Arik
Published: July 2002

TFIIH is a multifunctional RNA polymerase II general initiation
factor that includes two DNA helicases encoded by the
Xeroderma
pigmentosum
complementation group B (
XPB
) and D
(
XPD
) genes and a cyclin-dependent protein kinase
encoded by the
CDK7
gene. Previous studies have shown
that the TFIIH XPB DNA helicase plays critical roles not only in
transcription initiation, where it catalyzes ATP-dependent formation of
the open complex, but also in efficient promoter escape, where it
suppresses arrest of very early RNA polymerase II elongation
intermediates. In this report, we present evidence that ATP-dependent
TFIIH action in transcription initiation and promoter escape requires
distinct regions of the DNA template; these regions are well separated
from the promoter region unwound by the XPB DNA helicase and extend,
respectively, ≈23–39 and ≈39–50 bp downstream from the
transcriptional start site. Taken together, our findings bring to light
a role for promoter DNA in TFIIH action and are consistent with the
model that TFIIH translocates along promoter DNA ahead of the RNA
polymerase II elongation complex until polymerase has escaped the
promoter.
Negative selection of thymocytes expressing the D10 TCR
Authors: Sant'Angelo Derek B. , Janeway Charles A.
Published: July 2002

We have analyzed the patterns of positive and negative selection of thymocytes expressing the T cell antigen receptor (TCR) from the D10.G4.1 T cell clone. This TCR confers reactivity to several non-self MHC class II alleles with a remarkably broad range of avidities. Therefore, negative selection can be studied when induced by high-, intermediate-, or low-avidity interactions with endogenous peptide–MHC complexes, all within the same TCR transgenic system. These data directly demonstrate that MHC class II–peptide ligands that fail to activate mature T cells can promote negative selection of immature thymocytes. Additionally, we show that negative selection of thymocytes can occur at two distinct “time points” during development depending on the avidity of the TCR for the MHC–peptide complex. Finally, we show that the self-peptide repertoire plays a significant role in selection because alteration of the self-peptide repertoire by disruption of the H2-Ma gene drastically alters selection of D10 TCR-expressing thymocytes.
The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine
Authors: Crespo José L. , Powers Ted , Fowler Brian , Hall Michael N.
Published: July 2002

The essential, rapamycin-sensitive TOR kinases regulate a diverse set of cell growth-related readouts in response to nutrients. Thus, the yeast TOR proteins function as nutrient sensors, in particular as sensors of nitrogen and possibly carbon. However, the nutrient metabolite(s) that acts upstream of TOR is unknown. We investigated the role of glutamine, a preferred nitrogen source and a key intermediate in yeast nitrogen metabolism, as a possible regulator of TOR. We show that the glutamine synthetase inhibitor
l
-methionine sulfoximine (MSX) specifically provokes glutamine depletion in yeast cells. MSX-induced glutamine starvation caused nuclear localization and activation of the TOR-inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate glutamine synthesis. The MSX-induced nuclear localization of GLN3 required the TOR-controlled, type 2A-related phosphatase SIT4. Other TOR-controlled transcription factors, GAT1/NIL1, MSN2, MSN4, and an unknown factor involved in the expression of ribosomal protein genes, were not affected by glutamine starvation. These findings suggest that the TOR pathway senses glutamine. Furthermore, as glutamine starvation affects only a subset of TOR-controlled transcription factors, TOR appears to discriminate between different nutrient conditions to elicit a response appropriate to a given condition.
The candidate tumor suppressor gene,
RASSF1A
, from human chromosome 3p21.3 is involved in kidney tumorigenesis
Authors: Dreijerink Koen , Braga Eleonora , Kuzmin Igor , Geil Laura , Duh Fuh-Mei , Angeloni Debora , Zbar Berton , Lerman Michael I. , Stanbridge Eric J. , Minna John D. , Protopopov Alexei , Li Jingfeng , Kashuba Vladimir , Klein George , Zabarovsky Eugene R.
Published: July 2002

Clear cell-type renal cell carcinomas (clear RCC) are characterized almost universally by loss of heterozygosity on chromosome 3p, which usually involves any combination of three regions: 3p25-p26 (harboring the
VHL
gene), 3p12-p14.2 (containing the
FHIT
gene), and 3p21-p22, implying inactivation of the resident tumor-suppressor genes (TSGs). For the 3p21-p22 region, the affected TSGs remain, at present, unknown. Recently, the RAS association family 1 gene (isoform RASSF1A), located at 3p21.3, has been identified as a candidate lung and breast TSG. In this report, we demonstrate aberrant silencing by hypermethylation of RASSF1A in both
VHL
-caused clear RCC tumors and clear RCC without
VHL
inactivation. We found hypermethylation of RASSF1A's GC-rich putative promoter region in most of analyzed samples, including 39 of 43 primary tumors (91%). The promoter was methylated partially or completely in all 18 RCC cell lines analyzed. Methylation of the GC-rich putative RASSF1A promoter region and loss of transcription of the corresponding mRNA were related causally. RASSF1A expression was reactivated after treatment with 5-aza-2′-deoxycytidine. Forced expression of RASSF1A transcripts in KRC/Y, a renal carcinoma cell line containing a normal and expressed VHL gene, suppressed growth on plastic dishes and anchorage-independent colony formation in soft agar. Mutant RASSF1A had reduced growth suppression activity significantly. These data suggest that
RASSF1A
is the candidate renal TSG gene for the 3p21.3 region.
Functional communication between endogenous BRCA1 and its partner, BARD1, during
Xenopus laevis
development
Authors: Joukov Vladimir , Chen Junjie , Fox Edward A. , Green Jeremy B. A. , Livingston David M.
Published: July 2002

The breast and ovarian susceptibility protein 1 (BRCA1) heterodimerizes with its structural relative, the BRCA1-associated RING domain protein (BARD1), which may have tumor suppressing function in its own right. Both proteins have evolved from a common evolutionary ancestor, and both exist in
Xenopus
laevis
where, similar to their mammalian homologs, they form functional heterodimers. Depleting frog embryos of either BARD1 or BRCA1 led to similar and widely defective developmental phenotypes as well as depletion of the other polypeptide due to its decreased stability. Thus, each protein, in part, controls the abundance, stability, and function of the other, and these effects are heterodimerization-dependent. The interdependent nature of BRCA1 and BARD1 function supports the view that BARD1/BRCA1 heterodimers play a major role in breast and ovarian cancer suppression.
Retinal engineering: engrafted neural cell lines locate in appropriate layers.
Authors: Trisler D , Rutin J , Pessac B
Published: July 2002

A major question in central nervous system development, including the neuroretina, is whether migrating cells express cues to find their way and settle at specific locations. We have transplanted quail neuroretinal cell lines QNR/D, a putative amacrine or ganglion cell, and QNR/K2, a putative Müller cell into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina; in contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. These data show that QNR/D and QNR/K2 cell lines represent distinct neural cell types, suggesting that migrating neural cells express distinct address cues. Furthermore, our results raise the possibility that immortalized cell lines can be used for replacement of specific cell types and for the transport of genes to given locations in neuroretina.
Restricted structural gene polymorphism in the
Mycobacterium tuberculosis
complex indicates evolutionarily recent global dissemination
Authors: Sreevatsan Srinand , Pan Xi , Stockbauer Kathryn E. , Connell Nancy D. , Kreiswirth Barry N. , Whittam Thomas S. , Musser James M.
Published: July 2002

One-third of humans are infected with
Mycobacterium tuberculosis
, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that
M. tuberculosis
is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of
M. tuberculosis
were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with
M. bovis
, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS
6110
subtypes of this genetic group have IS
6110
integrated at the identical chromosomal insertion site, located between
dnaA
and
dnaN
in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.
Identification of a cellular receptor for subgroup E avian leukosis virus
Authors: Adkins Heather B. , Brojatsch Jürgen , Naughton John , Rolls Melissa M. , Pesola Jean M. , Young John A. T.
Published: July 2002

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.
Trajectory of DNA in the RNA polymerase II transcription preinitiation complex
Authors: Kim Tae-Kyung , Lagrange Thierry , Wang Yuh-Hwa , Griffith Jack D. , Reinberg Danny , Ebright Richard H.
Published: July 2002

By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, ≈240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of ≈50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.
Characterization and cell cycle regulation of the related human telomeric proteins Pin2 and TRF1 suggest a role in mitosis
Authors: Shen Minhui , Haggblom Candy , Vogt Marguerite , Hunter Tony , Lu Kun Ping
Published: July 2002

Telomeres are essential for preserving chromosome integrity during the cell cycle and have been specifically implicated in mitotic progression, but little is known about the signaling molecule(s) involved. The human telomeric repeat binding factor protein (TRF1) is shown to be important in regulating telomere length. However, nothing is known about its function and regulation during the cell cycle. The sequence of
PIN2
, one of three human genes (
PIN1
-
3
) we previously cloned whose products interact with the
Aspergillus
NIMA cell cycle regulatory protein kinase, reveals that it encodes a protein that is identical in sequence to TRF1 apart from an internal deletion of 20 amino acids; Pin2 and TRF1 may be derived from the same gene,
PIN2/TRF1
. However, in the cell Pin2 was found to be the major expressed product and to form homo- and heterodimers with TRF1; both dimers were localized at telomeres. Pin2 directly bound the human telomeric repeat DNA
in vitro
, and was localized to all telomeres uniformly in telomerase-positive cells. In contrast, in several cell lines that contain barely detectable telomerase activity, Pin2 was highly concentrated at only a few telomeres. Interestingly, the protein level of Pin2 was highly regulated during the cell cycle, being strikingly increased in G2+M and decreased in G1 cells. Moreover, overexpression of Pin2 resulted in an accumulation of HeLa cells in G2+M. These results indicate that Pin2 is the major human telomeric protein and is highly regulated during the cell cycle, with a possible role in mitosis. The results also suggest that Pin2/TRF1 may connect mitotic control to the telomere regulatory machinery whose deregulation has been implicated in cancer and aging.
Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton
Authors: Malloy Kirk D. , Holman Molly A. , Mitchell David , Detrich H. William
Published: July 2002

The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlate with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers.
Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase
Authors: Dean Antony M. , Golding G. Brian
Published: July 2002

Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC
1.1.1.42
) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of
Escherichia coli
isocitrate dehydrogenase and the distantly related
Thermus thermophilus
NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation.
Promoter-creating mutations in
Pseudomonas putida
: A model system for the study of mutation in starving bacteria
Authors: Kasak Lagle , Hõrak Rita , Kivisaar Maia
Published: July 2002

A novel experimental system to study mutation in starving bacteria was designed, relying on the activation of a promoterless phenol degradation operon of
Pseudomonas putida
. The Phe
+
(phenol-utilizing) mutants accumulated in the starving culture of
P. putida
in the presence of phenol but not in the absence of it. We ruled out the possibility that the absence of phenol eliminates Phe
+
mutants from the starving population. Sequence analysis of the Phe
+
mutants revealed that base substitutions, deletions, and insertion of Tn
4652
can result in creation of a sequence similar to the σ
70
-specific promoter consensus. One particular C → A transversion was predominant in the Phe
+
mutants that arose in the starving population under selection for phenol use. In contrast, various deletions were the most frequent Phe
+
mutants occurring in a culture growing without selection. The accumulation rate of the Phe
+
mutants on selective plates was found to be higher for bacteria plated from stationary-phase culture than that from exponentially growing cells. This suggests that some specific processes, occurring predominantly in stationary-phase cells, facilitate generation and/or fixation of such mutations.
Fertilization-independent seed development in
Arabidopsis thaliana
Authors: Chaudhury Abdul M. , Ming Luo , Miller Celia , Craig Stuart , Dennis Elizabeth S. , Peacock W. James
Published: July 2002

We report mutants in
Arabidopsis thaliana
(fertilization-independent seed:
fis
) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the
pistillata
phenotype. Although the
pistillata
(
pi
) mutant has short siliques devoid of seed, the
fis
mutants in the
pi
background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three
fis
mutations map to loci on three different chromosomes. In
fis1
and
fis2
seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When
FIS/fis
plants are pollinated by pollen from
FIS/FIS
plants, ≈50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (
FIS/FIS
). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (
FIS/fis
). In normal sexual reproduction, the products of the
FIS
genes are likely to play important regulatory roles in the development of seed after fertilization.
A new reporter cell line to monitor HIV infection and drug susceptibilityin vitro
Authors: Gervaix Alain , West Daniel , Leoni Lorenzo M. , Richman Douglas D. , Wong-Staal Flossie , Corbeil Jacques
Published: July 2002

Determination of HIV infectivityin vitroand its inhibition by antiretroviral drugs by monitoring reduction of production of p24 antigen is expensive and time consuming. Such assays also do not allow accurate quantitation of the number of infected cells over time. To develop a simple, rapid, and direct method for monitoring HIV infection, we generated a stable T-cell line (CEM) containing a plasmid encoding the green fluorescent protein (humanized S65T GFP) driven by the HIV-1 long terminal repeat. Clones were selected that displayed low constitutive background fluorescence, but a high level of GFP expression upon infection with HIV. HIV-1 infection induced a 100- to 1,000-fold increase in relative fluorescence of cells over 2 to 4 days as monitored by fluorescence microscopy, cytofluorimetry, and flow cytometry. Addition of inhibitors of reverse transcriptase, protease, and other targets at different multiplicities of infection permitted the accurate determination of drug susceptibility. This technique also permitted quantitation of infectivity of viral preparations by assessment of number of cells infected in the first round of infection. In conclusion, the CEM-GFP reporter cell line provides a simple, rapid, and direct method for monitoring HIV infectivity titers and antiretroviral drug susceptibility of syncytium-inducing strains.
MsPG3
, a
Medicago sativa
polygalacturonase gene expressed during the alfalfa–
Rhizobium meliloti
interaction
Authors: Muñoz Jose A. , Coronado Carmen , Pérez-Hormaeche Javier , Kondorosi Adam , Ratet Pascal , Palomares Antonio J.
Published: July 2002

Polygalacturonase (PG) is one of the most important enzymes associated with plant cell wall degradation. It has been proposed to participate in the early steps of the
Rhizobium
–legume interaction. We have identified two classes of cDNA fragments corresponding to two classes of PG genes in the
Medicago
genome. One of this class, represented by E
2
in
M. truncatula
and Pl
1
in
M. sativa
, seems to be related to previously characterized plant PG genes expressed in pollen. We have isolated the genomic clone containing the entire gene corresponding to the second class (E
3
). We showed that
MsPG3
is a single gene in the
Medicago
genome coding for PG. By reverse transcription-PCR,
MsPG3
expression was detected in roots 1 day after
Rhizobium
inoculation. The early induction of the
MsPG3
, as also seen by
in situ
hybridization experiments, supports its involvement in the early stages of the
Rhizobium
-legume infection process. In addition, by analyzing the expression of a
MsPG3
promoter-
gus
construct in
Vicia hirsuta
-transgenic root nodules, we showed that
MsPG3
was expressed in all cells of nodule primordia and in the cells of the invasion zone. By Northern blot,
MsPG3
transcripts are not detected in various
Medicago
tissues, indicating that the function of this gene is related closely to symbiosis. Thus, our results strongly suggest the involvement of
MsPG3
gene during meristem formation and/or in the infection process, probably by facilitating cell wall rearrangement, penetration of the bacteria through the root hair wall, or infection thread formation and release of bacteria in plant cells.
MsPG3
represents a class of PG genes, distinct from the pollen-specific genes, and it is the first pectic encoded enzyme demonstrated to be involved in
Rhizobium
-legume symbiosis.
Initial cash/asset ratio and asset prices: An experimental study
Authors: Caginalp Gunduz , Porter David , Smith Vernon
Published: July 2002

A series of experiments, in which nine participants trade an asset over 15 periods, test the hypothesis that an initial imbalance of asset/cash will influence the trading price over an extended time. Participants know at the outset that the asset or “stock” pays a single dividend with fixed expectation value at the end of the 15th period. In experiments with a greater total value of cash at the start, the mean prices during the trading periods are higher, compared with those with greater amount of asset, with a high degree of statistical significance. The difference is most significant at the outset and gradually tapers near the end of the experiment. The results are very surprising from a rational expectations and classical game theory perspective, because the possession of a large amount of cash does not lead to a simple motivation for a trader to bid excessively on a financial instrument. The gradual erosion of the difference toward the end of trading, however, suggests that fundamental value is approached belatedly, offering some consolation to the rational expectations theory. It also suggests that there is a time scale on which an evolution toward fundamental value occurs. The experimental results are qualitatively compatible with the price dynamics predicted by a system of differential equations based on asset flow. The results have broad implications for the marketing of securities, particularly initial and secondary public offerings, government bonds, etc., where excess supply has been conjectured to suppress prices.
A single binding site for dilysine retrieval motifs and p23 within the γ subunit of coatomer
Authors: Harter Cordula , Wieland Felix T.
Published: July 2002

Coatomer, the major component of the coat of COPI transport vesicles, binds both to the dilysine motif of resident membrane proteins of the endoplasmic reticulum and to the cytoplasmic domain of p23, a major type I membrane protein of COPI vesicles. Using a photocrosslinking approach, we find that under native conditions a peptide analogous to the cytoplasmic domain of p23 interacts with coatomer exclusively through its γ subunit and shares its binding site with a KKXX retrieval motif. However, upon dissociation of coatomer, interaction with various subunits, including an α-, β′-, ɛ-COP subcomplex, of the photoreactive peptide is observed. We suggest that, under physiological conditions, interaction of coatomer with both endoplasmic reticulum retrieval motifs and the cytoplasmic domain of p23 is mediated by γ-COP.
GroEL-GroES-mediated protein folding requires an intact central cavity
Authors: Wang Jue D. , Michelitsch Melissa D. , Weissman Jonathan S.
Published: July 2002

The chaperonin GroEL is an oligomeric double ring structure that, together with the cochaperonin GroES, assists protein folding. Biochemical analyses indicate that folding occurs in a cis ternary complex in which substrate is sequestered within the GroEL central cavity underneath GroES. Recently, however, studies of GroEL “minichaperones” containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. Here we compare directly the folding activity of minichaperones with that of the full GroEL-GroES system. In agreement with earlier studies, minichaperones assist folding of some proteins. However, this effect is observed only under conditions where substantial spontaneous folding is also observed and is indistinguishable from that resulting from addition of the nonchaperone protein α-casein. By contrast, the full GroE system efficiently promotes folding of several substrates under conditions where essentially no spontaneous folding is observed. These data argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.
Major histocompatibility complex variation associated with juvenile survival and parasite resistance in a large unmanaged ungulate population (Ovis aries L.)
Authors: Paterson S. , Wilson K. , Pemberton J. M.
Published: July 2002

Antagonistic coevolution between hosts and parasites has been proposed as a mechanism maintaining genetic diversity in both host and parasite populations. In particular, the high levels of genetic diversity widely observed at the major histocompatibility complex (MHC) of vertebrate hosts are consistent with the hypothesis of parasite-driven balancing selection acting to maintain MHC genetic diversity. To date, however, empirical evidence in support of this hypothesis, especially from natural populations, has been lacking. A large unmanaged population of Soay sheep (Ovis aries L.) is used to investigate associations between MHC variation, juvenile survival, and parasite resistance. We show in an unmanaged, nonhuman population that allelic variation within the MHC is significantly associated with differences in both juvenile survival and resistance to intestinal nematodes. Certain MHC alleles are associated with low survivorship probabilities and high levels of parasitism or vice versa. We conclude that parasites are likely to play a major role in the maintenance of MHC diversity in this population.
TRP2: A candidate transduction channel for mammalian pheromone sensory signaling
Authors: Liman Emily R. , Corey David P. , Dulac Catherine
Published: July 2002

The vomeronasal organ (VNO) of terrestrial vertebrates plays a key role in the detection of pheromones, chemicals released by animals that elicit stereotyped sexual and aggressive behaviors among conspecifics. Sensory transduction in the VNO appears unrelated to that in the vertebrate olfactory and visual systems: the putative pheromone receptors of the VNO are evolutionarily independent from the odorant receptors and, in contrast to vertebrate visual and olfactory transduction, vomeronasal transduction is unlikely to be mediated by cyclic-nucleotide-gated channels. We hypothesized that sensory transduction in the VNO might instead involve an ion channel of the transient receptor potential (TRP) family, members of which mediate cyclic-nucleotide-independent sensory responses in
Drosophila melanogaster
and
Caenorhabditis elegans
and play unknown functions in mammals. We have isolated a cDNA (rTRP2) from rat VNO encoding a protein of 885 amino acids that is equally distant from vertebrate and invertebrate TRP channels (10–30% amino acid identity). rTRP2 mRNA is exclusively expressed in VNO neurons, and the protein is highly localized to VNO sensory microvilli, the proposed site of pheromone sensory transduction. The absence of Ca
2+
stores in sensory microvilli suggests that, in contrast to a proposed mechanism of activation of mammalian TRP channels, but in accord with analysis of TRP function in
Drosophila
phototransduction, the gating of TRP2 is independent from the depletion of internal Ca
2+
stores. Thus, TRP2 is likely to participate in vomeronasal sensory transduction, which may share additional similarities with light-induced signaling in the
Drosophila
eye.
Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation
Authors: Arnoult Christophe , Kazam Imrana G. , Visconti Pablo E. , Kopf Gregory S. , Villaz Michel , Florman Harvey M.
Published: July 2002

Sperm adhesion to egg zonae pellucidae initiates sperm acrosome reactions, an exocytotic event that is an early step during fertilization. Previously, it was suggested that zona pellucida-evoked Ca
2+
entry into sperm through low voltage-activated Ca
2+
channels is an essential step in acrosome reactions, based on the inhibitory effects of Ca
2+
channel antagonists. However, analysis of this channel is limited by the inability to apply electrophysiological methods directly to sperm. In this report, optical methods of determining membrane potential and internal Ca
2+
levels were used to demonstrate that (
i
) contact with zonae pellucidae activates a transient Ca
2+
response in sperm that has a time course and antagonist sensitivity anticipated of low voltage-activated Ca
2+
channels; (
ii
) these channels are unavailable for opening in uncapacitated sperm because of voltage-dependent, steady state inactivation; (
iii
) membrane hyperpolarization during sperm capacitation is sufficient to recruit channels into a closed state, from which they are available for opening during fertilization; and (
iv
) channel conductance state may be a factor in determines the efficacy with which channel antagonists inhibit fertilization. This study provides evidence for the activation of sperm Ca
2+
channels during gamete adhesion and offers a mechanism that may account for aspects of the regulation of sperm fertility during capacitation through the control of channel availability. Finally, these results suggest that channel conductance state may be a central feature in the design of channel antagonists that inhibit sperm function.
Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-α-glucosidase
Authors: Amalfitano A. , McVie-Wylie A. J. , Hu H. , Dawson T. L. , Raben N. , Plotz P. , Chen Y. T.
Published: July 2002

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid α-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.
Functional characterization of B lymphocytes generatedin vitrofrom embryonic stem cells
Authors: Cho Sarah K. , Webber Travis D. , Carlyle James R. , Nakano Toru , Lewis Susanna M. , Zúñiga-Pflücker Juan Carlos
Published: July 2002

To study molecular events involved in B lymphocyte development and V(D)J rearrangement, we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here, we show that B lineage cells generatedin vitrofrom ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first, they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second, they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third, they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover, the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably, EAB cells possess functional V(D)J recombinase activity. In particular, the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation.
An invasion-associated
Salmonella
protein modulates the actin-bundling activity of plastin
Authors: Zhou Daoguo , Mooseker Mark S. , Galán Jorge E.
Published: July 2002

The entry of
Salmonella typhimurium
into nonphagocytic cells requires a panel of bacterial effector proteins that are delivered to the host cell via a type III secretion system. These proteins modulate host–cell signal-transduction pathways and the actin cytoskeleton to induce membrane ruffling and bacterial internalization. One of these bacterial effectors, termed SipA, is an actin-binding protein that is required for efficient
Salmonella
entry into host cells. We report here that SipA forms a complex with T-plastin on bacterial infection. Formation of such a complex, which requires the presence of F-actin, results in a marked increase in the actin-bundling activity of T-plastin. We also report that T-plastin is recruited to
S. typhimurium
-induced membrane ruffles by a CDC42-dependent signaling process and is required for bacterial entry. We propose that modulation of the actin-bundling activity of T-plastin by SipA results in the stabilization of the actin filaments at the point of bacterial–host cell contact, which leads to more efficient
Salmonella
internalization.
Inhibition of T cell proliferation by selective block of Ca
2+
-activated K
+
channels
Authors: Jensen Bo Skaaning , Ødum Niels , Jørgensen Nanna Koschmieder , Christophersen Palle , Olesen Søren-Peter
Published: July 2002

T lymphocytes express a plethora of distinct ion channels that participate in the control of calcium homeostasis and signal transduction. Potassium channels play a critical role in the modulation of T cell calcium signaling, and the significance of the voltage-dependent K channel, Kv1.3, is well established. The recent cloning of the Ca
2+
-activated, intermediate-conductance K
+
channel (IK channel) has enabled a detailed investigation of the role of this highly Ca
2+
-sensitive K
+
channel in the calcium signaling and subsequent regulation of T cell proliferation. The role IK channels play in T cell activation and proliferation has been investigated by using various blockers of IK channels. The Ca
2+
-activated K
+
current in human T cells is shown by the whole-cell voltage-clamp technique to be highly sensitive to clotrimazole, charybdotoxin, and nitrendipine, but not to ketoconazole. Clotrimazole, nitrendipine, and charybdotoxin block T cell activation induced by signals that elicit a rise in intracellular Ca
2+
—e.g., phytohemagglutinin, Con A, and antigens such as
Candida albicans
and tetanus toxin in a dose-dependent manner. The release of IFN-γ from activated T cells is also inhibited after block of IK channels by clotrimazole. Clotrimazole and cyclosporin A act synergistically to inhibit T cell proliferation, which confirms that block of IK channels affects the process downstream from T cell receptor activation. We suggest that IK channels constitute another target for immune suppression.
Key residues revealed in a major conformational epitope of the U1-70K protein
Authors: Welin Henriksson Elisabet , Wahren-Herlenius Marie , Lundberg Ingrid , Mellquist Evi , Pettersson Ingvar
Published: July 2002

Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, we have used diversely mutated recombinantDrosophila melanogaster70K proteins as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Our results reveal that amino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119–126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains, and tertiary structure modeling indicates that the residues 119–126 are situated at easily accessible positions in the end of an α-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact.
Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2
Authors: Munroe Donald G. , Gupta Ashwani K. , Kooshesh Fatemeh , Vyas Tejal B. , Rizkalla Geihan , Wang Hong , Demchyshyn Lidia , Yang Zhi-Jie , Kamboj Rajender K. , Chen Hongyun , McCallum Kirk , Sumner-Smith Martin , Drucker Daniel J. , Crivici Anna
Published: July 2002

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived
peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates
intestinal growth and up-regulates villus height in the small
intestine, concomitant with increased crypt cell proliferation and
decreased enterocyte apoptosis. Moreover, GLP-2 prevents
intestinal hypoplasia resulting from total parenteral nutrition.
However, the mechanism underlying these actions has remained unclear.
Here we report the cloning and characterization of cDNAs encoding rat
and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor
superfamily member expressed in the gut and closely related to the
glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome
17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1
or related peptides, with increased cAMP production (EC
50
=
0.58 nM) and displayed saturable high-affinity radioligand binding
(
K
d
= 0.57 nM), which could be displaced by
synthetic rat GLP-2 (
K
i
= 0.06 nM). GLP-2
analogs that activated GLP-2R signal transduction
in
vitro
displayed intestinotrophic activity
in
vivo
. These results strongly suggest that GLP-2, like glucagon
and GLP-1, exerts its actions through a distinct and specific novel
receptor expressed in its principal target tissue, the gastrointestinal
tract.
Novel gene conversion between X-Y homologues located in the nonrecombining region of the Y chromosome in Felidae (Mammalia)
Authors: Pecon Slattery Jill , Sanner-Wachter Leslie , O'Brien Stephen J.
Published: July 2002

Genes located on the mammalian Y chromosome outside of the pseudoautosomal region do not recombine with those on the X and are predicted to either undergo selection for male function or gradually degenerate because of an accumulation of deleterious mutations. Here, phylogenetic analyses of X-Y homologues,
Zfx
and
Zfy
, among 26 felid species indicate two ancestral episodes of directed genetic exchange (ectopic gene conversion) from X to Y: once during the evolution of pallas cat and once in a common predecessor of ocelot lineage species. Replacement of the more rapidly evolving Y homologue with the evolutionarily constrained X copy may represent a mechanism for adaptive editing of functional genes on the nonrecombining region of the mammalian Y chromosome.
Suppressed T helper 2 immunity and prolonged survival of a nematode parasite in protein-malnourished mice
Authors: Ing Rebecca , Su Zhong , Scott Marilyn E. , Koski Kristine G.
Published: July 2002

Protein malnutrition may increase susceptibility to gastrointestinal parasitic infections, possibly as a result of impaired intestinal and/or systemic T helper 2 (Th2) effector responses induced by down-regulation of Th2 cytokines and/or up-regulation of Th1 cytokines. To test this hypothesis, female BALB/c mice (n= 18/diet) were fed a control (24%), marginal (7%), or deficient (3%) protein diet and given a challenge infection withHeligmosomoides polygyrus. The 3% mice had higher worm burdens at 1, 2, and 4 weeks postchallenge infection (pci), lower increases in serum IgE, reduced intestinal eosinophilia, and depressed mucosal mast cell proliferation and activation at 1–2 weeks pci. To determine whether these suppressed effector responses resulted from altered spleen and mesenteric lymph node (MLN) cytokine production, cells were restimulatedin vitrowith parasite antigen and cytokine concentrations were measured. Deficient MLN cells secreted significantly less IL-4 and more IFN-γ at 1–2 weeks pci than did control MLN cells. Deficient spleen cells also secreted more IFN-γ at 2 weeks pci compared with control spleen cells. From reverse transcription-PCR analyses, the 3% mice also had lower IL-4 mRNA level in spleen and MLN at 1–2 weeks pci. Our study supports the hypothesis that protein malnutrition increases the survival of a nematode parasite by decreasing gut-associated IL-4 (Th2) and increasing IFN-γ (Th1) within 2 weeks pci, leading to reduced intestinal and systemic Th2 effector responses.
Enhanced learning after genetic overexpression of a brain growth protein
Authors: Routtenberg Aryeh , Cantallops Isabel , Zaffuto Sal , Serrano Peter , Namgung Uk
Published: July 2002

Ramón y Cajal proposed 100 years ago that memory formation requires the growth of nerve cell processes. One-half century later, Hebb suggested that growth of presynaptic axons and postsynaptic dendrites consequent to coactivity in these synaptic elements was essential for such information storage. In the past 25 years, candidate growth genes have been implicated in learning processes, but it has not been demonstrated that they in fact enhance them. Here, we show that genetic overexpression of the growth-associated protein GAP-43, the axonal protein kinase C substrate, dramatically enhanced learning and long-term potentiation in transgenic mice. If the overexpressed GAP-43 was mutated by a Ser → Ala substitution to preclude its phosphorylation by protein kinase C, then no learning enhancement was found. These findings provide evidence that a growth-related gene regulates learning and memory and suggest an unheralded target, the GAP-43 phosphorylation site, for enhancing cognitive ability.
Genetic analysis of a bacterial genetic exchange element: The gene transfer agent of
Rhodobacter capsulatus
Authors: Lang Andrew S. , Beatty J. T.
Published: July 2002

An unusual system of genetic exchange exists in the purple
nonsulfur bacterium
Rhodobacter capsulatus
. DNA
transmission is mediated by a small bacteriophage-like particle called
the gene transfer agent (GTA) that transfers random 4.5-kb segments of
the producing cell's genome to recipient cells, where allelic
replacement occurs. This paper presents the results of gene cloning,
analysis, and mutagenesis experiments that show that GTA resembles a
defective prophage related to bacteriophages from diverse genera of
bacteria, which has been adopted by
R. capsulatus
for
genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to
constitute part of a sensor kinase/response regulator signaling
pathway that is required for expression of GTA structural genes. This
signaling pathway controls growth-phase-dependent regulation of GTA
gene messages, yielding maximal gene expression in the stationary
phase. We suggest that GTA is an ancient prophage remnant that has
evolved in concert with the bacterial genome, resulting in a genetic
exchange process controlled by the bacterial cell.