bluuebunny/crossref_metadata_2025 huggingface dataset:
A cell number-counting factor regulates the cytoskeleton and cell motility in
Dictyostelium
Authors: Tang Lei , Gao Tong , McCollum Catherine , Jang Wonhee , Vicker Michael G. , Ammann Robin R. , Gomer Richard H.
Published: July 2002

Little is known about how a morphogenetic rearrangement of a tissue is affected by individual cells. Starving
Dictyostelium discoideum
cells aggregate to form dendritic streams, which then break up into groups of ≈2 × 10
4
cells. Cell number is sensed at this developmental stage by using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF indicates that there is a large number of cells, which then causes the aggregation stream to break up. Computer simulations indicated that stream breakup could be caused by CF decreasing cell–cell adhesion and/or increasing cell motility, and we observed that CF does indeed decrease cell–cell adhesion. We find here that CF increases cell motility. In
Dictyostelium
, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and the ABP-120 actin-crosslinking protein. Partially inhibiting motility by using drugs that interfere with actin polymerization reduces stream dissipation, resulting in fewer stream breaks and thus larger groups. CF also potentiates the phosphorylation and redistribution of myosin while repressing its basal level of assembly. The computer simulations indicated that a narrower distribution of group sizes results when a secreted factor modulates both adhesion and motility. CF thus seems to induce the morphogenesis of streams into evenly sized groups by increasing actin polymerization, ABP-120 levels, and myosin phosphorylation and decreasing adhesion and myosin polymerization.
Mutation rates in mammalian genomes
Authors: Kumar Sudhir , Subramanian Sankar
Published: July 2002

Knowledge of the rate of point mutation is of fundamental importance, because mutations are a vital source of genetic novelty and a significant cause of human diseases. Currently, mutation rate is thought to vary many fold among genes within a genome and among lineages in mammals. We have conducted a computational analysis of 5,669 genes (17,208 sequences) from species representing major groups of placental mammals to characterize the extent of mutation rate differences among genes in a genome and among diverse mammalian lineages. We find that mutation rate is approximately constant per year and largely similar among genes. Similarity of mutation rates among lineages with vastly different generation lengths and physiological attributes points to a much greater contribution of replication-independent mutational processes to the overall mutation rate. Our results suggest that the average mammalian genome mutation rate is 2.2 × 10−9per base pair per year, which provides further opportunities for estimating species and population divergence times by using molecular clocks.
Compartmentation protects trypanosomes from the dangerous design of glycolysis
Authors: Bakker Barbara M. , Mensonides Femke I. C. , Teusink Bas , van Hoek Pim , Michels Paul A. M. , Westerhoff Hans V.
Published: July 2002

Unlike in other organisms, in trypanosomes and other Kinetoplastida the larger part of glycolysis takes place in a specialized organelle, called the glycosome. At present it is impossible to remove the glycosome without changing much of the rest of the cell. It would seem impossible, therefore, to assess the metabolic consequences of this compartmentation. Therefore, we here develop a computer experimentation approach, which we call computational cell biology. A validated molecular kinetic computer replica was built of glycolysis in the parasite
Trypanosoma brucei.
Removing the glycosome membrane in that replica had little effect on the steady-state flux, which argues against the prevalent speculation that glycosomes serve to increase flux by concentrating the enzymes. Removal of the membrane did cause (
i
) the sugar phosphates to rise to unphysiologically high levels, which must have pathological effects, and (
ii
) a failure to recover from glucose deprivation. We explain these effects on the basis of the biochemical organization of the glycosome. We conclude (
i
) that the glycosome protects trypanosomes from the negative side effects of the “turbo” structure of glycolysis and (
ii
) that computer experimentation based on solid molecular data is a powerful tool to address questions that are not, or not yet, accessible to experimentation.
The complete genome of hyperthermophile
Methanopyrus kandleri AV19
and monophyly of archaeal methanogens
Authors: Slesarev Alexei I. , Mezhevaya Katja V. , Makarova Kira S. , Polushin Nikolai N. , Shcherbinina Olga V. , Shakhova Vera V. , Belova Galina I. , Aravind L. , Natale Darren A. , Rogozin Igor B. , Tatusov Roman L. , Wolf Yuri I. , Stetter Karl O. , Malykh Andrei G. , Koonin Eugene V. , Kozyavkin Sergei A.
Published: July 2002

We have determined the complete 1,694,969-nt sequence of the GC-rich genome of
Methanopyrus kandleri
by using a whole direct genome sequencing approach. This approach is based on unlinking of genomic DNA with the ThermoFidelase version of
M. kandleri
topoisomerase V and cycle sequencing directed by 2′-modified oligonucleotides (Fimers). Sequencing redundancy (3.3×) was sufficient to assemble the genome with less than one error per 40 kb. Using a combination of sequence database searches and coding potential prediction, 1,692 protein-coding genes and 39 genes for structural RNAs were identified.
M. kandleri
proteins show an unusually high content of negatively charged amino acids, which might be an adaptation to the high intracellular salinity. Previous phylogenetic analysis of 16S RNA suggested that
M. kandleri
belonged to a very deep branch, close to the root of the archaeal tree. However, genome comparisons indicate that, in both trees constructed using concatenated alignments of ribosomal proteins and trees based on gene content,
M. kandleri
consistently groups with other archaeal methanogens.
M. kandleri
shares the set of genes implicated in methanogenesis and, in part, its operon organization with
Methanococcus jannaschii
and
Methanothermobacter thermoautotrophicum
. These findings indicate that archaeal methanogens are monophyletic. A distinctive feature of
M. kandleri
is the paucity of proteins involved in signaling and regulation of gene expression. Also,
M. kandleri
appears to have fewer genes acquired via lateral transfer than other archaea. These features might reflect the extreme habitat of this organism.
Dominant negative down-regulation of endotoxin-induced tumor necrosis factor α production byLpsd/Ran
Authors: Yuan Quan , Zhao Fangping , Chung Siu-Wah , Fan Peidong , Sultzer Barnet M. , Kan Y. W. , Wong Peter M. C.
Published: July 2002

We recently showed that adenoviral transfer and expression of theLpsd/Ran gene isolated from endotoxin-resistant C3H/HeJ mice could protect endotoxin-sensitive mice from endotoxic shock. Elevation of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), is thought to be essential for the development of septic shock. To investigate the extent to whichLpsd/Ranaffects TNF-α production, we transduced primary macrophages from endotoxin-sensitive and -resistant mice with adenoviral vectors expressing the wild-type and the mutantLps/RancDNAs and other control genes, and compared the amount of TNF-α produced by these various transduced macrophages. Successful transfer and expression ofLpsd/RancDNA in endotoxin-sensitive C3H/HeOuJ macrophages reduced TNF-α production upon lipopolysaccharide (LPS) stimulation, as compared with macrophages transduced with vectors expressing the wild-typeLpsn/RancDNA, the green fluorescent protein gene, or thelacZgene. On the other hand, successful transfer and expression of the wild-typeLpsn/RancDNA in primary macrophages from endotoxin-resistant C3H/HeJ mice failed to induce TNF-α production to any significant extent unless a very high LPS concentration was used. Given our previous demonstration that Lpsn/Ran functions effectively in restoring LPS responsiveness in B cells from C3H/HeJ mice, we conclude thatLps/Ranis involved in a CD14-independent signal transduction pathway. This dominant negative down-regulation byLpsd/Ranon TNF-α production by macrophages and probably other innate immune responses may be key to the development of an effective gene therapy for endotoxic or septic shock.
Recent improvements in prediction of protein structure by global optimization of a potential energy function
Authors: Pillardy Jarosław , Czaplewski Cezary , Liwo Adam , Lee Jooyoung , Ripoll Daniel R. , Kaźmierkiewicz Rajmund , Ołdziej Stanisław , Wedemeyer William J. , Gibson Kenneth D. , Arnautova Yelena A. , Saunders Jeff , Ye Yuan-Jie , Scheraga Harold A.
Published: July 2002

Recent improvements of a hierarchical
ab initio
or
de novo
approach for predicting both α and β
structures of proteins are described. The united-residue energy
function used in this procedure includes multibody interactions from a
cumulant expansion of the free energy of polypeptide chains, with their
relative weights determined by Z-score optimization. The critical
initial stage of the hierarchical procedure involves a search of
conformational space by the conformational space annealing (CSA)
method, followed by optimization of an all-atom model. The procedure
was assessed in a recent blind test of protein structure prediction
(CASP4). The resulting lowest-energy structures of the target proteins
(ranging in size from 70 to 244 residues) agreed with the experimental
structures in many respects. The entire experimental structure of a
cyclic α-helical protein of 70 residues was predicted to within 4.3
Å α-carbon (C
α
) rms deviation (rmsd) whereas, for
other α-helical proteins, fragments of roughly 60 residues were
predicted to within 6.0 Å C
α
rmsd. Whereas β
structures can now be predicted with the new procedure, the success
rate for α/β- and β-proteins is lower than that for
α-proteins at present. For the β portions of α/β
structures, the C
α
rmsd's are less than 6.0 Å for
contiguous fragments of 30–40 residues; for one target, three
fragments (of length 10, 23, and 28 residues, respectively) formed a
compact part of the tertiary structure with a C
α
rmsd
less than 6.0 Å. Overall, these results constitute an important step
toward the
ab initio
prediction of protein structure
solely
from the amino acid sequence.
Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy
in vivo
Authors: Rothermel Beverly A. , McKinsey Timothy A. , Vega Rick B. , Nicol Rebekka L. , Mammen Pradeep , Yang John , Antos Christopher L. , Shelton John M. , Bassel-Duby Rhonda , Olson Eric N. , Williams R. Sanders
Published: July 2002

Signaling events controlled by calcineurin promote cardiac
hypertrophy, but the degree to which such pathways are required to
transduce the effects of various hypertrophic stimuli remains
uncertain. In particular, the administration of immunosuppressive drugs
that inhibit calcineurin has inconsistent effects in blocking cardiac
hypertrophy in various animal models. As an alternative approach to
inhibiting calcineurin in the hearts of intact animals, transgenic mice
were engineered to overexpress a human cDNA encoding the
calcineurin-binding protein, myocyte-enriched calcineurin-interacting
protein-1 (hMCIP1) under control of the cardiac-specific, α-myosin
heavy chain promoter (α-MHC). In unstressed mice, forced expression
of hMCIP1 resulted in a 5–10% decline in cardiac mass relative to
wild-type littermates, but otherwise produced no apparent structural or
functional abnormalities. However, cardiac-specific expression of
hMCIP1 inhibited cardiac hypertrophy, reinduction of fetal gene
expression, and progression to dilated cardiomyopathy that otherwise
result from expression of a constitutively active form of calcineurin.
Expression of the hMCIP1 transgene also inhibited hypertrophic
responses to β-adrenergic receptor stimulation or exercise training.
These results demonstrate that levels of hMCIP1 producing no apparent
deleterious effects in cells of the normal heart are sufficient to
inhibit several forms of cardiac hypertrophy, and suggest an important
role for calcineurin signaling in diverse forms of cardiac hypertrophy.
The future development of measures to increase expression or activity
of MCIP proteins selectively within the heart may have clinical value
for prevention of heart failure.
Paleoclimate and Amerindians: Evidence from stable isotopes and atmospheric circulation
Authors: Lovvorn Marjorie Brooks , Frison George C. , Tieszen Larry L.
Published: July 2002

Two Amerindian demographic shifts are attributed to climate change
in the northwest plains of North America: at ≈11,000 calendar years
before present (yr BP), Amerindian culture apparently split into
foothills–mountains vs. plains biomes; and from 8,000–5,000 yr BP,
scarce archaeological sites on the open plains suggest emigration
during xeric “Altithermal” conditions. We reconstructed
paleoclimates from stable isotopes in prehistoric bison bone and
relations between weather and fractions of C
4
plants in
forage. Further, we developed a climate-change model that synthesized
stable isotope, existing qualitative evidence (e.g., palynological,
erosional), and global climate mechanisms affecting this midlatitude
region. Our isotope data indicate significant warming from ≈12,400 to
11,900 yr BP, supporting climate-driven cultural separation. However,
isotope evidence of apparently wet, warm conditions at 7,300 yr BP
refutes emigration to avoid xeric conditions. Scarcity of
archaeological sites is best explained by rapid climate fluctuations
after catastrophic draining of the Laurentide Lakes, which disrupted
North Atlantic Deep Water production and subsequently altered monsoonal
inputs to the open plains.
Morphological abnormalities in the brains of estrogen receptor β knockout mice
Authors: Wang Ling , Andersson Sandra , Warner Margaret , Gustafsson Jan-Åke
Published: July 2002

Estrogen receptor β (ERβ) is expressed at high levels in both
neurons and glial cells of the central nervous system. The development
of ERβ knockout (BERKO) mice has provided a model to study the
function of this nuclear receptor in the brain. We have found that the
brains of BERKO mice show several morphological abnormalities. There is
a regional neuronal hypocellularity in the brain, with a severe
neuronal deficit in the somatosensory cortex, especially layers II,
III, IV, and V, and a remarkable proliferation of astroglial cells in
the limbic system but not in the cortex. These abnormalities are
evident as early as 2 mo of age in BERKO mice. As BERKO mice age, the
neuronal deficit becomes more pronounced, and, by 2 yr of age, there is
degeneration of neuronal cell bodies throughout the brain. This is
particularly evident in the substantia nigra. We conclude that ERβ is
necessary for neuronal survival and speculate that this gene could have
an important influence on the development of degenerative diseases of
the central nervous system, such as Alzheimer's disease and
Parkinson's disease, as well as those resulting from trauma and stroke
in the brain.
Early statistical detection of anthrax outbreaks by tracking over-the-counter medication sales
Authors: Goldenberg Anna , Shmueli Galit , Caruana Richard A. , Fienberg Stephen E.
Published: July 2002

The recent series of anthrax attacks has reinforced the importance of biosurveillance systems for the timely detection of epidemics. This paper describes a statistical framework for monitoring grocery data to detect a large-scale but localized bioterrorism attack. Our system illustrates the potential of data sources that may be more timely than traditional medical and public health data. The system includes several layers, each customized to grocery data and tuned to finding footprints of an epidemic. We also propose an evaluation methodology that is suitable in the absence of data on large-scale bioterrorist attacks and disease outbreaks.
Discrimination of MHC-derived odors by untrained mice is consistent with divergence in peptide-binding region residues
Authors: Carroll Lara S. , Penn Dustin J. , Potts Wayne K.
Published: July 2002

Genes of the major histocompatibility complex (MHC) play a central role in immune recognition, yet they also influence the odor of individuals. Mice can be trained to distinguish odors mediated byclassicalMHC loci; however, training can introduce confounding behavioral artifacts. This study demonstrates that mice can distinguish some, but not all, naturally occurring allelic variants atclassicalMHC loci without prior training. This result suggests that MHC-disassortative mating preferences might operate by means of small MHC-based odor differences, and could therefore contribute to diversifying selection acting on MHC loci. Here we show that odors of two MHC mutant mouse strains (bm1andbm3) can be distinguished, even after genetic background is controlled by intercrossing strains. These two strains differ by five amino acids, three of which are predicted to chemically contact peptides bound to the peptide-binding region (PBR), the site of antigen presentation for T cell recognition. However, the odors of neitherbm1norbm3were distinguished from their parental B6 haplotype after randomizing genomic background, despite discrimination of pure-bred B6 andbm1strain odors. These combined results suggest that (i) there may be an MHC odor discrimination threshold based on divergence in PBR residues, providing a more logical pattern of MHC-based odor discrimination than found in previous training studies, where discrimination ability was not correlated with PBR divergence; and (ii) additional (non-MHC) mutations that influence odor have accumulated in these strains during the 100 generations of divergence between pure B6 andbm1strains.
Murine retroviruses activate B cells via interaction with toll-like receptor 4
Authors: Rassa John C. , Meyers Jennifer L. , Zhang Yuanming , Kudaravalli Rama , Ross Susan R.
Published: July 2002

Although most retroviruses require activated cells as their targets for infection, it is not known how this is achievedin vivo. A candidate protein for the activation of B cells by either mouse mammary tumor virus (MMTV) or murine leukemia virus is the toll-like receptor 4 (TLR4), a component of the innate immune system. MMTV caused B cell activation in C3H/HeN mice but not in C3H/HeJ or BALB/c (C.C3HTlr4lps-d) congenic mice, both of which have a mutantTLR4gene. This activation was independent of viral gene expression, because it occurred after treatment of MMTV with ultraviolet light or 2,2′-dithiodipyridine and in azidothymidine-treated mice. Nuclear extracts prepared from the lymphocytes of MMTV-injected C3H/HeN but not C3H/HeJ mice showed increased nuclear factor κB activity. Additionally, the MMTV- and Moloney murine leukemia virus envelope proteins coimmunoprecipitated with TLR4 when expressed in 293T cells. The MMTV receptor failed to coimmunoprecipitate with TLR4, suggesting that MMTV/TLR4 interaction is independent of virus attachment and fusion. These results identify retroviral proteins that interact with a mammalian toll receptor and show that direct activation by such viruses may initiatein vivoinfection pathways.
Profilin I is essential for cell survival and cell division in early mouse development
Authors: Witke Walter , Sutherland James D. , Sharpe Arlene , Arai Maya , Kwiatkowski David J.
Published: July 2002

Profilins are thought to play a central role in the regulation of
de novo
actin assembly by preventing spontaneous actin
polymerization through the binding of actin monomers, and the adding of
monomeric actin to the barbed actin-filament ends. Other cellular
functions of profilin in membrane trafficking and lipid based signaling
are also likely. Binding of profilins to signaling molecules such as
Arp2/3 complex, Mena, VASP, N
-
WASP, dynamin I, and
others, further implicates profilin and actin as regulators of diverse
motile activities. In mouse, two profilins are expressed from two
distinct genes. Profilin I is expressed at high levels in all tissues
and throughout development, whereas profilin II is expressed in
neuronal cells. To examine the function of profilin I
in
vivo
, we generated a null profilin I
(
pfn1
ko
) allele in mice. Homozygous
pfn1
ko/ko
mice are not viable.
Pfn1
ko/ko
embryos died as early as the
two-cell stage, and no
pfn1
ko/ko
blastocysts
were detectable. Adult
pfn1
ko/wt
mice show a
50% reduction in profilin I expression with no apparent impairment of
cell function. However,
pfn1
ko/wt
embryos
have reduced survival during embryogenesis compared with wild type.
Although weakly expressed in early embryos, profilin II cannot
compensate for lack of profilin I. Our results indicate that mouse
profilin I is an essential protein that has dosage-dependent effects on
cell division and survival during embryogenesis.
Wolbachiadensity and virulence attenuation after transfer into a novel host
Authors: McGraw E. A. , Merritt D. J. , Droller J. N. , O'Neill S. L.
Published: July 2002

The factors that control replication rate of the intracellular bacteriumWolbachia pipientisin its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain ofWolbachia that is known to over-replicate and shorten the lifespan of itsDrosophila melanogasterhost, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachiaassociation. After transinfection (the transfer of aWolbachiastrain into a different species) of the over-replicatingWolbachia popcornstrain fromD. melanogastertoDrosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect inD. simulansattenuated only slightly and was comparable to that induced inD. melanogaster. This study reveals a strong host involvement inWolbachiareplication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.
Adenoviral gene transfer of
Caenorhabditis elegans n
−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells
Authors: Kang Zhao B. , Ge Yinlin , Chen Zhihong , Cluette-Brown Joanne , Laposata Michael , Leaf Alexander , Kang Jing X.
Published: July 2002

Omega−3 polyunsaturated fatty acids (PUFAs) are essential
components required for normal cellular function and have been shown to
exert many preventive and therapeutic actions. The amount of
n
−3 PUFAs is insufficient in most Western people,
whereas the level of
n
−6 PUFAs is relatively too high,
with an
n
−6/
n
−3 ratio of >18. These
two classes of PUFAs are metabolically and functionally distinct and
often have important opposing physiological functions; their balance is
important for homeostasis and normal development. Elevating tissue
concentrations of
n
−3 PUFAs in mammals relies on
chronic dietary intake of fat rich in
n
−3 PUFAs,
because mammalian cells lack enzymatic activities necessary either to
synthesize the precursor of
n
−3 PUFAs or to convert
n
−6 to
n
−3 PUFAs. Here we report that
adenovirus-mediated introduction of the
Caenorhabditis elegans
fat-1
gene encoding an
n
−3 fatty acid
desaturase into mammalian cells can quickly and effectively elevate the
cellular
n
−3 PUFA contents and dramatically balance the
ratio of
n
−6/
n
−3 PUFAs. Heterologous
expression of the
fat-1
gene in rat cardiac myocytes
rendered cells capable of converting various
n
−6 PUFAs
to the corresponding
n
−3 PUFAs, and changed the
n
−6/
n
−3 ratio from about 15:1 to 1:1.
In addition, an eicosanoid derived from
n
−6 PUFA (i.e.,
arachidonic acid) was reduced significantly in the transgenic cells.
This study demonstrates an effective approach to modifying fatty acid
composition of mammalian cells and also provides a basis for potential
applications of this gene transfer in experimental and clinical
settings.
Benthic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment
Authors: Edgcomb Virginia P. , Kysela David T. , Teske Andreas , de Vera Gomez Alvin , Sogin Mitchell L.
Published: July 2002

Molecular microbial ecology studies have revealed remarkable prokaryotic diversity in extreme hydrothermal marine environments. There are no comparable reports of culture-independent surveys of eukaryotic life in warm, anoxic marine sediments. By using sequence comparisons of PCR-amplified small subunit ribosomal RNAs, we characterized eukaryotic diversity in hydrothermal vent environments of Guaymas Basin in the Gulf of California. Many sequences from these anoxic sediments and the overlaying seawater represent previously uncharacterized protists, including early branching eukaryotic lineages or extended diversity within described taxa. At least two mechanisms, with overlapping consequences, account for the eukaryotic community structure of this environment. The adaptation to anoxic environments is evidenced by specific affinity of environmental sequences to aerotolerant anaerobic species in molecular trees. This pattern is superimposed against a background of widely distributed aerophilic and aerotolerant protists, some of which may migrate into and survive in the sediment whereas others (e.g., phototrophs) are simply deposited by sedimentary processes. In contrast, bacterial populations in these sediments are primarily characteristic of anoxic, reduced, hydrocarbon-rich sedimentary habitats.
Mammalian germ-line transgenesis by transposition
Authors: Dupuy Adam J. , Clark Karl , Carlson Corey M. , Fritz Sabine , Davidson Ann E. , Markley Karra M. , Finley Ken , Fletcher Colin F. , Ekker Stephen C. , Hackett Perry B. , Horn Sandra , Largaespada David A.
Published: July 2002

Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called
Sleeping Beauty
in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.
Virus-assisted loading of polymer nanocontainer
Authors: Graff Alexandra , Sauer Marc , Van Gelder Patrick , Meier Wolfgang
Published: July 2002

We present a DNA-containing polymeric nanocontainer using the self-assembled superstructure of amphiphilic block copolymers in aqueous solutions. To demonstrate that DNA translocation is possible across a completely synthetic block copolymer membrane, we have used a phage transfection strategy as a DNA-transfer model system. For this purpose the bacterial channel forming protein LamB was reconstituted in ABA-triblock copolymer vesicles. The outer membrane protein LamB is a specific transporter for maltodextrins but also serves as a receptor for λ phage to trigger the ejection of λ phage DNA. We demonstrate that the functionality of the LamB protein is fully preserved despite the artificial surrounding. This leads to a type of polymeric vehicle for DNA that could be useful for gene therapy.
Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor
Authors: Xu Weidong , Angelis Konstantina , Danielpour David , Haddad Maher M. , Bischof Oliver , Campisi Judith , Stavnezer Ed , Medrano Estela E.
Published: July 2002

The c-
ski
protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β-mediated growth inhibition in a prostate-derived epithelial cell line.
RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells
Authors: Yu Jenn-Yah , DeRuiter Stacy L. , Turner David L.
Published: July 2002

Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by
in vitro
transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific β-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.
Interrogating protein interaction networks through structural biology
Authors: Aloy Patrick , Russell Robert B.
Published: July 2002

Protein–protein interactions are central to most biological processes. Although much recent effort has been put into methods to identify interacting partners, there has been a limited focus on how these interactions compare with those known from three-dimensional (3D) structures. Because comparison of protein interactions often involves considering homologous, but not identical, proteins, a key issue is whether proteins that are homologous to an interacting pair will interact in the same way, or interact at all. Accordingly, we describe a method to test putative interactions on complexes of known 3D structure. Given a 3D complex and alignments of homologues of the interacting proteins, we assess the fit of any possible interacting pair on the complex by using empirical potentials. For studies of interacting protein families that show different specificities, the method provides a ranking of interacting pairs useful for prioritizing experiments. We evaluate the method on interacting families of proteins with multiple complex structures. We then consider the fibroblast growth factor/receptor system and explore the intersection between complexes of known structure and interactions proposed between yeast proteins by methods such as two-hybrids. We provide confirmation for several interactions, in addition to suggesting molecular details of how they occur.
Reconciling paleodistribution models and comparative phylogeography in the Wet Tropics rainforest land snailGnarosophia bellendenkerensis(Brazier 1875)
Authors: Hugall Andrew , Moritz Craig , Moussalli Adnan , Stanisic John
Published: July 2002

Comparative phylogeography has proved useful for investigating biological responses to past climate change and is strongest when combined with extrinsic hypotheses derived from the fossil record or geology. However, the rarity of species with sufficient, spatially explicit fossil evidence restricts the application of this method. Here, we develop an alternative approach in which spatial models of predicted species distributions under serial paleoclimates are compared with a molecular phylogeography, in this case for a snail endemic to the rainforests of North Queensland, Australia. We also compare the phylogeography of the snail to those from several endemic vertebrates and use consilience across all of these approaches to enhance biogeographical inference for this rainforest fauna. The snail mtDNA phylogeography is consistent with predictions from paleoclimate modeling in relation to the location and size of climatic refugia through the late Pleistocene-Holocene and broad patterns of extinction and recolonization. There is general agreement between quantitative estimates of population expansion from sequence data (using likelihood and coalescent methods) vs. distributional modeling. The snail phylogeography represents a composite of both common and idiosyncratic patterns seen among vertebrates, reflecting the geographically finer scale of persistence and subdivision in the snail. In general, this multifaceted approach, combining spatially explicit paleoclimatological models and comparative phylogeography, provides a powerful approach to locating historical refugia and understanding species' responses to them.
Automated parallel DNA sequencing on multiple channel microchips
Authors: Liu Shaorong , Ren Hongji , Gao Qiufeng , Roach David J. , Loder Robert T. , Armstrong Thomas M. , Mao Qinglu , Blaga Iuliu , Barker David L. , Jovanovich Stevan B.
Published: July 2002

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect
.
We present experimental evidence consistent with the predictions of the theory.
neuralized
is essential for a subset of Notch pathway-dependent cell fate decisions during
Drosophila
eye development
Authors: Lai Eric C. , Rubin Gerald M.
Published: July 2002

neuralized
(
neur
) is a
neurogenic mutant of
Drosophila
in which many signaling
events mediated by the Notch (N) receptor are disrupted. Here, we
analyze the role of
neur
during eye development.
Neur
is required in a cell-autonomous fashion to
restrict R8 and other photoreceptor fates and is involved in lateral
inhibition of interommatidial bristles but is not required for
induction of the cone cell fate. The latter contrasts with the absolute
requirement for
Suppressor of Hairless
and the
Enhancer of split-
Complex for cone cell induction. Using
gain-of-function experiments, we further demonstrate that ectopic
wild-type and truncated Neur proteins can interfere with multiple
N-controlled aspects of eye development, including both
neur
-dependent and
neur
-independent
processes.
Regulation of the stem cell leukemia (
SCL
) gene: A tale of two fishes
Authors: Barton Linda M. , Göttgens Berthold , Gering Martin , Gilbert James G. R. , Grafham Darren , Rogers Jane , Bentley David , Patient Roger , Green Anthony R.
Published: July 2002

The stem cell leukemia (
SCL
) gene encodes a
tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal
role in hemopoiesis and vasculogenesis. Several enhancers have been
identified within the murine
SCL
locus that direct
reporter gene expression to subdomains of the normal
SCL
expression pattern, and long-range sequence comparisons of the human
and murine
SCL
loci have identified additional candidate
enhancers. To facilitate the characterization of regulatory elements,
we have sequenced and analyzed 33 kb of the
SCL
genomic
locus from the pufferfish
Fugu rubripes
, a species with
a highly compact genome. Although the pattern of
SCL
expression is highly conserved from mammals to teleost fish, the genes
flanking pufferfish
SCL
were unrelated to those known to
flank both avian and mammalian
SCL
genes. These data
suggest that
SCL
regulatory elements are confined to the
region between the upstream and downstream flanking genes, a region of
65 kb in human and 8.5 kb in pufferfish. Consistent with this
hypothesis, the entire 33-kb pufferfish
SCL
locus
directed appropriate expression to hemopoietic and neural tissue in
transgenic zebrafish embryos, as did a 10.4-kb fragment containing the
SCL
gene and extending to the 5′ and 3′ flanking genes.
These results demonstrate the power of combining the compact genome of
the pufferfish with the advantages that zebrafish provide for studies
of gene regulation during development. Furthermore, the pufferfish
SCL
locus provides a powerful tool for the manipulation
of hemopoiesis and vasculogenesis
in vivo
.
Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α
1S
II-III loop
Authors: Wilkens Christina M. , Kasielke Nicole , Flucher Bernhard E. , Beam Kurt G. , Grabner Manfred
Published: July 2002

The II-III loop of the skeletal muscle dihydropyridine receptor
(DHPR) α
1S
subunit is responsible for
bidirectional-signaling interactions with the ryanodine receptor
(RyR1): transmitting an orthograde, excitation–contraction (EC)
coupling signal to RyR1 and receiving a retrograde, current-enhancing
signal from RyR1. Previously, several reports argued for the importance
of two distinct regions of the skeletal II-III loop (residues
R681–L690 and residues L720–Q765, respectively), claiming for each a
key function in DHPR–RyR1 communication. To address whether residues
720–765 of the II-III loop are sufficient to enable skeletal-type
(Ca
2+
entry-independent) EC coupling and retrograde
interaction with RyR1, we constructed a green fluorescent protein
(GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR
sequence except for a II-III loop (L) from the DHPR of the house fly,
Musca domestica
(M). The
Musca
II-III
loop (75% dissimilarity to α
1S
) has no similarity to
α
1S
in the regions R681–L690 and L720–Q765. GFP-SkLM
expressed in dysgenic myotubes (which lack endogenous
α
1S
subunits) was unable to restore EC coupling and
displayed strongly reduced Ca
2+
current densities despite
normal surface expression levels and correct triad targeting
(colocalization with RyR1). Introducing rabbit α
1S
residues L720–L764 into the
Musca
II-III loop of
GFP-SkLM (substitution for
Musca
DHPR residues
E724–T755) completely restored bidirectional coupling, indicating its
dependence on α
1S
loop residues 720–764 but its
independence from other regions of the loop. Thus, 45
α
1S
-residues embedded in a very dissimilar background are
sufficient to restore bidirectional coupling, indicating that these
residues may be a site of a protein–protein interaction required for
bidirectional coupling.
Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca
2+
channel
Authors: Ahern Chris A. , Arikkath Jyothi , Vallejo Paola , Gurnett Christina A. , Powers Patricia A. , Campbell Kevin P. , Coronado Roberto
Published: July 2002

To investigate the molecular basis of the voltage sensor that
triggers excitation–contraction (EC) coupling, the four-domain pore
subunit of the dihydropyridine receptor (DHPR) was cut in the
cytoplasmic linker between domains II and III. cDNAs for the I-II
domain (α1S 1–670) and the III-IV domain (α1S 701-1873) were
expressed in dysgenic α1S-null myotubes. Coexpression of the two
fragments resulted in complete recovery of DHPR intramembrane charge
movement and voltage-evoked Ca
2+
transients. When fragments
were expressed separately, EC coupling was not recovered. However,
charge movement was detected in the I-II domain expressed alone.
Compared with I-II and III-IV together, the charge movement in the I-II
domain accounted for about half of the total charge
(
Q
max
= 3 ± 0.23 vs. 5.4 ±
0.76 fC/pF, respectively), and the half-activation potential for
charge movement was significantly more negative
(
V
1/2
= 0.2 ± 3.5 vs. 22 ±
3.4 mV, respectively). Thus, interactions between the four internal
domains of the pore subunit in the assembled DHPR profoundly affect the
voltage dependence of intramembrane charge movement. We also tested a
two-domain I-II construct of the neuronal α1A Ca
2+
channel. The neuronal I-II domain recovered charge movements like those
of the skeletal I-II domain but could not assist the skeletal III-IV
domain in the recovery of EC coupling. The results demonstrate that a
functional voltage sensor capable of triggering EC coupling in skeletal
myotubes can be recovered by the expression of complementary fragments
of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing
properties of the α1A I-II domain suggest that this
hemi-Ca
2+
channel could be relevant to neuronal function.
Exploring the DNA-binding specificities of zinc fingers with DNA microarrays
Authors: Bulyk Martha L. , Huang Xiaohua , Choo Yen , Church George M.
Published: July 2002

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.
Selection for
in vivo
regulators of bacterial virulence
Authors: Lee Sang Ho , Butler Susan M. , Camilli Andrew
Published: July 2002

We devised a noninvasive genetic selection strategy to
identify positive regulators of bacterial virulence genes during actual
infection of an intact animal host. This strategy combines random
mutagenesis with a switch-like reporter of transcription that confers
antibiotic resistance in the off state and sensitivity in the on state.
Application of this technology to the human intestinal pathogen
Vibrio cholerae
identified several regulators of cholera
toxin and a central virulence gene regulator that are operative during
infection. These regulators function in chemotaxis, signaling pathways,
transport across the cell envelope, biosynthesis, and adherence. We
show that phenotypes that appear genetically independent in cell
culture become interrelated in the host milieu.
The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL-ARNT fusion
Authors: Salomon-Nguyen Florence , Della-Valle Véronique , Mauchauffé Martine , Busson-Le Coniat Maryvonne , Ghysdael Jacques , Berger Roland , Bernard Olivier A.
Published: July 2002

The
TEL
/
ETV6
gene is located at 12p13 and encodes a member of the ETS family of transcription factors. Translocated ETS leukemia (TEL) is frequently involved in chromosomal translocations in human malignancies, usually resulting in the expression of fusion proteins between the amino-terminal part of TEL and either unrelated transcription factors or protein tyrosine kinases. We have characterized a t(1;12)(q21;p13) translocation in an acute myeloblastic leukemia (AML-M2). At the protein level, the untranslocated TEL copy and, as a result of the t(1;12) translocation, a fusion protein between TEL and essentially all of aryl hydrocarbon receptor nuclear translocator (ARNT) are expressed. The involvement of ARNT in human leukemogenesis has not been previously described. The ARNT protein belongs to a subfamily of the “basic region helix–loop–helix” (bHLH) protein that shares an additional region of similarity called the PAS (Per, ARNT, SIM) domain. ARNT is the central partner of several heterodimeric transcription factors, including those containing the aryl hydrocarbon (dioxin) receptor (AhR) and the hypoxia-inducible factor 1α (HIF1α). Our results show that the TEL-ARNT fusion protein is the crucial product of the translocation and suggest that interference with the activity of AhR or HIF1α can contribute to leukemogenesis.
A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge
Authors: Deliyannis Georgia , Boyle Jefferey S. , Brady Jamie L. , Brown Lorena E. , Lew Andrew M.
Published: July 2002

Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs
in vivo
). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2–24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.