bluuebunny/crossref_metadata_2025 huggingface dataset:
The human SWI/SNF-B chromatin-remodeling complex is related to yeast Rsc and localizes at kinetochores of mitotic chromosomes
Authors: Xue Yutong , Canman Julie C. , Lee Cheol Soon , Nie Zuqin , Yang Dafeng , Moreno G. Tony , Young Mary K. , Salmon E. D. , Wang Weidong
Published: July 2002

The SWI/SNF family of chromatin-remodeling complexes facilitates
gene expression by helping transcription factors gain access to their
targets in chromatin. SWI/SNF and Rsc are distinctive members of this
family from yeast. They have similar protein components and catalytic
activities but differ in biological function. Rsc is required for cell
cycle progression through mitosis, whereas SWI/SNF is not. Human
complexes of this family have also been identified, which have often
been considered related to yeast SWI/SNF. However, all human subunits
identified to date are equally similar to components of both SWI/SNF
and Rsc, leaving open the possibility that some or all of the human
complexes are rather related to Rsc. Here, we present evidence that the
previously identified human SWI/SNF-B complex is indeed of the Rsc
type. It contains six components conserved in both Rsc and SWI/SNF.
Importantly, it has a unique subunit, BAF180, that harbors a
distinctive set of structural motifs characteristic of three components
of Rsc. Of the two mammalian ATPases known to be related to those in
the yeast complexes, human SWI/SNF-B contains only the homolog that
functions like Rsc during cell growth. Immunofluorescence studies with
a BAF180 antibody revealed that SWI/SNF-B localizes at the
kinetochores of chromosomes during mitosis. Our data suggest that
SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling
complexes conserved from yeast to human, and could participate in cell
division at kinetochores of mitotic chromosomes.
Escaping the stem cell compartment: Sustained UVB exposure allows
p53
-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations
Authors: Zhang Wengeng , Remenyik Eva , Zelterman Daniel , Brash Douglas E. , Wikonkal Norbert M.
Published: July 2002

Once mutated, a single cell must expand into a clone before becoming significant for carcinogenesis. The forces driving clonal expansion and the obstacles that must be overcome are poorly understood. In a genetic mechanism, acquiring a second mutation conferring a proliferative advantage would enable the cell to expand autonomously. If carcinogen exposure instead induced a physiological change, clonal expansion would require the carcinogen's continued presence. To determine which is the case, we studied microscopic clones of keratinocytes mutated in the
p53
tumor suppressor gene. Carcinogen exposure was controlled by irradiating mice with 280–320 nm UV radiation (UVB), sunlight's principal carcinogenic component; expansion of mutant clones was observed in epidermal sheets.
p53
-mutant clones grew only during chronic UVB exposure. Therefore, clonal expansion was not triggered by a proliferative mutation but was instead continually driven by UVB. Unexpectedly, the clone size distribution showed periodicity with maxima at estimated intervals of 16 ± 6 cells, the size of the epidermal proliferating unit in murine dorsal skin. In the absence of UVB, rare “imprisoned clones” increased in cell number without increasing in area. We conclude that: stem cell compartments act as physical barriers to clonal expansion of a
p53
-mutant keratinocyte; a rate-limiting step in clonal expansion is the colonization of an adjacent compartment; and sustained UVB enables the
p53
-mutant keratinocyte to colonize without incurring an additional mutation.
BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia
Authors: Tanner Stephan M. , Austin Jamie L. , Leone Gustavo , Rush Laura J. , Plass Christoph , Heinonen Kristiina , Mrózek Krzysztof , Sill Heinz , Knuutila Sakari , Kolitz Jonathan E. , Archer Kellie J. , Caligiuri Michael A. , Bloomfield Clara D. , de la Chapelle Albert
Published: July 2002

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3,
BAALC
(
b
rain
a
nd
a
cute
l
eukemia,
c
ytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized
BAALC
on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior.
In vitro
studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different
BAALC
transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing
BAALC
show distinctly poor prognosis, pointing to a key role of the
BAALC
products in leukemia. Our data suggest that
BAALC
is a gene implicated in both neuroectodermal and hematopoietic cell functions.
Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA
Authors: Neeleman Lyda , Olsthoorn René C. L. , Linthorst Huub J. M. , Bol John F.
Published: July 2002

On entering a host cell, positive-strand RNA virus genomes have to
serve as messenger for the translation of viral proteins. Efficient
translation of cellular messengers requires interactions between
initiation factors bound to the 5′-cap structure and the poly(A)
binding protein bound to the 3′-poly(A) tail. Initiation of
infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV)
and viruses from the genus
Ilarvirus
requires binding of
a few molecules of coat protein (CP) to the 3′ end of the
nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs
can be initiated by addition of the subgenomic messenger for CP, RNA 4.
We report here that extension of the AMV RNAs with a poly(A) tail of 40
to 80 A-residues permitted initiation of infection independently of CP
or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1
relieved an apparent bottleneck in the translation of the viral RNAs.
Translation of RNA 4 in plant protoplasts was autocatalytically
stimulated by its encoded CP. Mutations that interfered with CP binding
to the 3′ end of viral RNAs reduced translation of RNA 4 to
undetectable levels. Possibly, CP of AMV and ilarviruses stimulates
translation of viral RNAs by acting as a functional analogue of poly(A)
binding protein or other cellular proteins.
Epitope tagging of chromosomal genes in
Salmonella
Authors: Uzzau Sergio , Figueroa-Bossi Nara , Rubino Salvatore , Bossi Lionello
Published: July 2002

We have developed a simple and efficient procedure for adding an
epitope-encoding tail to one or more genes of interest in the bacterial
chromosome. The procedure is a modification of the gene
replacement method of Datsenko and Wanner [Datsenko, K. A. &
Wanner, B. L. (2000)
Proc. Natl. Acad. Sci. USA
97,
6640–6645]. A DNA module that begins with the epitope-encoding
sequence and includes a selectable marker is amplified by PCR with
primers that carry extensions (as short as 36 nt) homologous to the
last portion of the targeted gene and to a region downstream from it.
Transformation of a strain expressing bacteriophage λ
red
functions yields recombinants carrying the targeted
gene fused to the epitope-encoding sequence. The resulting
C-terminal-tagged protein can be identified by standard
immuno-detection techniques. In an initial application of the method,
we have added the sequences encoding the FLAG and 3xFLAG and influenza
virus hemagglutinin epitopes to various genes of
Salmonella
enterica
serovar Typhimurium, including putative and
established pathogenic determinants present in prophage genomes.
Epitope fusion proteins were detected in bacteria growing
in
vitro
, tissue culture cells, and infected mouse tissues. This
work identified a prophage locus specifically expressed in
bacteria growing intracellularly. The procedure described here should
be applicable to a wide variety of Gram-negative bacteria and is
particularly suited for the study of intracellular pathogens.
Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol.
Authors: Tsai S C , Adamik R , Moss J , Vaughan M
Published: July 2002

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.
Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.
Authors: Ha T , Enderle T , Ogletree D F , Chemla D S , Selvin P R , Weiss S
Published: July 2002

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.
Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.
Authors: Oldham S M , Clark G J , Gangarosa L M , Coffey R J , Der C J
Published: July 2002

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.
The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.
Authors: De Smet C , De Backer O , Faraoni I , Lurquin C , Brasseur F , Boon T
Published: July 2002

Human gene MAGE-1 encodes tumor-specific antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. This gene is expressed in a significant proportion of tumors of various histological types, but not in normal tissues except male germ-line cells. We reported previously that reporter genes driven by the MAGE-1 promoter are active not only in the tumor cell lines that express MAGE-1 but also in those that do not. This suggests that the critical factor causing the activation of MAGE-1 in certain tumors is not the presence of the appropriate transcription factors. The two major MAGE-1 promoter elements have an Ets binding site, which contains a CpG dinucleotide. We report here that these CpG are demethylated in the tumor cell lines that express MAGE-1, and are methylated in those that do not express the gene. Methylation of these CpG inhibits the binding of transcription factors, as seen by mobility shift assay. Treatment with the demethylating agent 5-aza-2'-deoxycytidine activated gene MAGE-1 not only in tumor cell lines but also in primary fibroblasts. Finally, the overall level of CpG methylation was evaluated in 20 different tumor cell lines. It was inversely correlated with the expression of MAGE-1. We conclude that the activation of MAGE-1 in cancer cells is due to the demethylation of the promoter. This appears to be a consequence of a genome-wide demethylation process that occurs in many cancers and is correlated with tumor progression.
Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.
Authors: Hochachka P W , Buck L T , Doll C J , Land S C
Published: July 2002

We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal-transduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine.
A carbonic anhydrase from the nacreous layer in oyster pearls.
Authors: Miyamoto H , Miyashita T , Okushima M , Nakano S , Morita T , Matsushiro A
Published: July 2002

It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.
Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.
Authors: Kriwacki R W , Hengst L , Tennant L , Reed S I , Wright P E
Published: July 2002

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.
A genetic selection for Caenorhabditis elegans synaptic transmission mutants.
Authors: Miller K G , Alfonso A , Nguyen M , Crowell J A , Johnson C D , Rand J B
Published: July 2002

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.
RGS-r, a retinal specific RGS protein, binds an intermediate conformation of transducin and enhances recycling
Authors: Chen Ching-Kang , Wieland Thomas , Simon Melvin I.
Published: July 2002

G proteins regulate intracellular signaling by coupling a cycle of
guanine nucleotide binding and hydrolysis to transient changes of
cellular functions. The mechanisms that control the recycling of
transducin, the “pace-setting” G protein that regulates mammalian
phototransduction, are unclear. We show that a novel retinal specific
RGS-motif protein specifically binds to an intermediate conformation
involved in GTP hydrolysis by transducin and accelerates phosphate
release and the recycling of transducin. This specific interaction
further rationalizes the kinetics of the phototransduction cascade and
provides a general hypothesis to explain the mechanism of interaction
of RGS proteins with other G proteins.
Double strand breaks at the
HIS2
recombination hot spot in
Saccharomyces cerevisiae
Authors: Bullard Steven A. , Kim Sangkyu , Galbraith Anne M. , Malone Robert E.
Published: July 2002

Double strand breaks (DSBs) have been found at several meiotic
recombination hot spots in
Saccharomyces cerevisiae
;
more global studies have found that they occur at many places along
several yeast chromosomes during meiosis. Indeed, the number of breaks
found is consistent with the number of recombination events predicted
from the genetic map. We have previously demonstrated that the
HIS2
gene is a recombination hot spot, exhibiting a high
frequency of gene conversion and associated crossing over. This paper
shows that DSBs occur in meiosis at a site in the coding region and at
a site downstream of the
HIS2
gene and that the DSBs are
dependent upon genes required for recombination. The frequency of DSBs
at
HIS2
increases when the gene conversion frequency is
increased by alterations in the DNA around
HIS2
, and
vice versa
. A deletion that increases both DSBs and
conversion can stimulate both when heterozygous; that is, it is
semidominant and acts to stimulate DSBs in trans. These data are
consistent with the view that homologous chromosomes associate with
each other before the formation of the DSBs.
Frequent clones of p53-mutated keratinocytes in normal human skin
Authors: Jonason Alan S. , Kunala Subrahmanyam , Price Gary J. , Restifo Richard J. , Spinelli Henry M. , Persing John A. , Leffell David J. , Tarone Robert E. , Brash Douglas E.
Published: July 2002

The multiple genetic hit model of cancer predicts that normal
individuals should have stable populations of cancer-prone, but
noncancerous, mutant cells awaiting further genetic hits. We report
that whole-mount preparations of human skin contain clonal patches of
p53-mutated keratinocytes, arising from the dermal–epidermal junction
and from hair follicles. These clones, 60–3000 cells in size, are
present at frequencies exceeding 40 cells per cm
2
and
together involve as much as 4% of the epidermis. In sun-exposed skin,
clones are both more frequent and larger than in sun-shielded skin. We
conclude that, in addition to being a tumorigenic mutagen, sunlight
acts as a tumor promoter by favoring the clonal expansion of
p53-mutated cells. These combined actions of sunlight result in normal
individuals carrying a substantial burden of keratinocytes predisposed
to cancer.
HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription
Authors: Rundlett Stephen E. , Carmen Andrew A. , Kobayashi Ryuji , Bavykin Sergei , Turner Bryan M. , Grunstein Michael
Published: July 2002

Increased histone acetylation has been correlated with increased
transcription, and regions of heterochromatin are generally
hypoacetylated. In investigating the cause-and-effect relationship
between histone acetylation and gene activity, we have characterized
two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is
an ≈350-kDa complex that is highly sensitive to the deacetylase
inhibitor trichostatin A. Histone deacetylase-B (HDB) is an ≈600-kDa
complex that is much less sensitive to trichostatin A. The HDA1 protein
(a subunit of the HDA activity) shares sequence similarity to RPD3, a
factor required for optimal transcription of certain yeast genes. RPD3
is associated with the HDB activity.
HDA1
also shares
similarity to three new open reading frames in yeast, designated
HOS1
,
HOS2
, and
HOS3
. We
find that both
hda1
and
rpd3
deletions
increase acetylation levels
in vivo
at all sites
examined in both core histones H3 and H4, with
rpd3
deletions having a greater impact on histone H4 lysine positions 5 and
12. Surprisingly, both
hda1
and
rpd3
deletions increase repression at telomeric loci, which resemble
heterochromatin with
rpd3
having a greater effect. In
addition,
rpd3
deletions retard full induction of the
PHO5
promoter fused to the reporter
lacZ
.
These data demonstrate that histone acetylation state has a role in
regulating both heterochromatic silencing and regulated gene
expression.
A synthetic
cryIC
gene, encoding a
Bacillus thuringiensis
δ-endotoxin, confers
Spodoptera
resistance in alfalfa and tobacco
Authors: Strizhov Nicolai , Keller Menachem , Mathur Jaideep , Koncz-Kálmán Zsuzsanna , Bosch Dirk , Prudovsky Evgenia , Schell Jeff , Sneh Baruch , Koncz Csaba , Zilberstein Aviah
Published: July 2002

Spodoptera
species, representing widespread
polyphagous insect pests, are resistant to
Bacillus
thuringiensis
δ-endotoxins used thus far as insecticides in
transgenic plants. Here we describe the chemical synthesis of a
cryIC
gene by a novel template directed ligation–PCR
method. This simple and economical method to construct large synthetic
genes can be used when routine resynthesis of genes is required.
Chemically phosphorylated adjacent oligonucleotides of the gene to be
synthesized are assembled and ligated on a single-stranded, partially
homologous template derived from a wild-type gene (
cryIC
in our case) by a thermostable
Pfu
DNA ligase using
repeated cycles of melting, annealing, and ligation. The resulting
synthetic DNA strands are selectively amplified by PCR with short
specific flanking primers that are complementary only to the new
synthetic DNA. Optimized expression of the synthetic
cryIC
gene in alfalfa and tobacco results in the
production of 0.01–0.2% of total soluble proteins as CryIC toxin and
provides protection against the Egyptian cotton leafworm
(
Spodoptera littoralis
) and the beet armyworm
(
Spodoptera exigua
). To facilitate selection and
breeding of
Spodoptera
-resistant plants, the
cryIC
gene was linked to a
pat
gene,
conferring resistance to the herbicide BASTA.
Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants.
Authors: Gromeier M , Alexander L , Wimmer E
Published: July 2002

Neuropathogenicity of poliovirus can be attenuated by mutations in the internal ribosomal entry site (IRES) within the 5' nontranslated region of its genome. The Sabin vaccine strains used in prevention of poliomyelitis carry such mutations in their IRES elements. In addition, mutations within the structural and nonstructural proteins of Sabin strains may equally contribute to the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis and, at a very low rate, they can cause poliomyelitis in vaccine recipients. The elimination of the neurocytopathic phenotype was achieved through the exchange of the entire poliovirus IRES with its counterpart from human rhinovirus type 2 without affecting growth properties in nonneuronal cells. The attenuating effect of the human rhinovirus type 2 IRES within the context of a poliovirus genome has been mapped to the 3' portion of this genetic element.
Identification of nitric oxide synthase as a protective locus against tuberculosis
Authors: MacMicking John D. , North Robert J. , LaCourse Ron , Mudgett John S. , Shah Shrenik K. , Nathan Carl F.
Published: July 2002

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primaryMycobacterium tuberculosisinfection using mice homozygous for a disruptedNOS2allele. NOS2−/−mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowedMycobacterium tuberculosisto replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus,NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration ofN6-(1-iminoethyl)-l-lysine. Together these findings identifyNOS2as a critical host gene for tuberculostasis.
Comparison of parametric and nonparametric methods to map oligogenes by linkage
Authors: Liò P. , Morton N. E.
Published: July 2002

A sample of 95 sib pairs affected with insulin-dependent diabetes and typed with their normal parents for 28 markers on chromosome 6 has been analyzed by several methods. When appropriate parameters are efficiently estimated, a parametric model is equivalent to the β model, which is superior to nonparametric alternatives both in single point tests (as found previously) and in multipoint tests. Theory is given for meta-analysis combined with allelic association, and problems that may be associated with errors of map location and/or marker typing are identified. Reducing by multipoint analysis the number of association tests in a dense map can give a 3-fold reduction in the critical lod, and therefore in the cost of positional cloning.
Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus
Authors: Herzog Roland W. , Hagstrom J. Nathan , Kung Szu-Hoo , Tai Shing Jen , Wilson James M. , Fisher Krishna J. , High Katherine A.
Published: July 2002

We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (10
12
–10
13
vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 1 mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200–350 ng/ml) of F.IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.
A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2
Authors: Vicente-Carbajosa Jesus , Moose Stephen P. , Parsons Ronald L. , Schmidt Robert J.
Published: July 2002

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys
2
-Cys
2
zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts
in vitro
with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.
The hotspot conversion paradox and the evolution of meiotic recombination
Authors: Boulton Alan , Myers Richard S. , Redfield Rosemary J.
Published: July 2002

Studies of meiotic recombination have revealed an evolutionary paradox. Molecular and genetic analysis has shown that crossing over initiates at specific sites called hotspots, by a recombinational-repair mechanism in which the initiating hotspot is replaced by a copy of its homolog. We have used computer simulations of large populations to show that this mechanism causes active hotspot alleles to be rapidly replaced by inactive alleles, which arise by rare mutation and increase by recombination-associated conversion. Additional simulations solidified the paradox by showing that the known benefits of recombination appear inadequate to maintain its mechanism. Neither the benefits of accurate segregation nor those of recombining flanking genes were sufficient to preserve active alleles in the face of conversion. A partial resolution to this paradox was obtained by introducing into the model an additional, nonmeiotic function for the sites that initiate recombination, consistent with the observed association of hotspots with functional sites in chromatin. Provided selection for this function was sufficiently strong, active hotspots were able to persist in spite of frequent conversion to inactive alleles. However, this explanation is unsatisfactory for two reasons. First, it is unlikely to apply to obligately sexual species, because observed crossover frequencies imply maintenance of many hotspots per genome, and the viability selection needed to preserve these would drive the species to extinction. Second, it fails to explain why such a genetically costly mechanism of recombination has been maintained over evolutionary time. Thus the paradox persists and is likely to be resolved only by significant changes to the commonly accepted mechanism of crossing over.
Frequency of HLA allele-specific peptide motifs in HIV-1 proteins correlates with the allele’s association with relative rates of disease progression after HIV-1 infection
Authors: Nelson George W. , Kaslow Richard , Mann Dean L.
Published: July 2002

An HLA allele-specific cytotoxic T lymphocyte response is thought to influence the rate of disease progression in HIV-1-infected individuals. In a prior study of 139 HIV-1-infected homosexual men, we identified HLA class I alleles and observed an association of specific alleles with different relative hazards for progression to AIDS. Seeking an explanation for this association, we searched HIV-1 protein sequences to determine the number of peptides matching motifs defined by combinations of specific amino acids reported to bind 16 class I alleles. Analyzing complete sequences of 12 clade B HIV isolates, we determined the number of allele motifs that were conserved (occurring in all 12 isolates) and nonconserved (occurring in only one isolate), as well as the average number of allele motifs per isolate. We found significant correlations with an allele’s association with disease progression for counts of conserved motifs in gag (R = 0.73;P= 0.002), pol (R = 0.58,P= 0.024), gp120 (R = 0.78,P= 0.00056), and total viral protein sequences (R = 0.67,P= 0.0058) and also for counts of nonconserved motifs in gag (R = 0.62,P= 0.013), pol (R = 0.74,P= 0.0017), gp41 (R = 0.52,P= 0.046), and total viral protein (R = 0.71,P= 0.0033). We also found significant correlations for the average number of motifs per isolate for gag, pol, gp120, and total viral protein. This study provides a plausible functional explanation for the observed association of different HLA alleles with variable rates of disease progression.
Fetal mouse selenophosphate synthetase 2 (SPS2): Characterization of the cysteine mutant form overproduced in a baculovirus–insect cell system
Authors: Kim Ick Young , Guimarães M. Jorge , Zlotnik Albert , Bazan J. Fernando , Stadtman Thressa C.
Published: July 2002

A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the
Escherichia coli
selenophosphate synthetase gene. Unlike the
E. coli
enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the
E. coli
enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus–insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide-dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.
Analysis of a peptide hormone–receptor interaction in the yeast two-hybrid system
Authors: Zhu Jianwei , Kahn C. Ronald
Published: July 2002

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a β-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 → His, Glu-9 → Val, Arg-37 → Gly, and Met-59 → Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 → Phe and Ile-43 → Phe), and two gave no detectable signal (Leu-14 → Arg and Glu-46 → Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 → Pro) that allowed for a strong IGF-1–receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.
Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis
Authors: Parkkila Seppo , Waheed Abdul , Britton Robert S. , Bacon Bruce R. , Zhou Xiao Yan , Tomatsu Shunji , Fleming Robert E. , Sly William S.
Published: July 2002

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to β
2
-microglobulin (β
2
M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrin-bound iron is normally transported to the fetus via receptor-mediated endocytosis. Western blot analyses show that the HFE protein is associated with β
2
M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein/β
2
M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal/fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload.
Selection of peptides that functionally replace a zinc finger in the Sp1 transcription factor by using a yeast combinatorial library
Authors: Cheng Xiaojun , Boyer Jennifer L. , Juliano R. L.
Published: July 2002

We have developed a strategy for the identification of peptides
able to functionally replace a zinc finger domain in a transcription
factor. This strategy could have important ramifications for basic
research on gene regulation and for the development of therapeutic
agents. In this study in yeast, we expressed chimeric proteins that
included a random peptide combinatorial library in association with two
zinc finger domains and a transactivating domain. The library was
screened for chimeric proteins capable of activating transcription from
a target sequence in the upstream regulatory regions of selectable or
reporter genes. In a screen of approximately 1.5 ×
10
7
transformants we identified 30 chimeric proteins that
exhibited transcriptional activation, some of which were able to
discriminate between wild-type and mutant DNA targets. Chimeric library
proteins expressed as glutathione
S
-transferase fusions
bound to double-stranded oligonucleotides containing the target
sequence, suggesting that the chimeras bind directly to DNA.
Surprisingly, none of the peptides identified resembled a zinc finger
or other well-known transcription factor DNA binding domain.
Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL
Authors: Goldberg Matthew S. , Zhang Jing , Sondek Stacey , Matthews C. Robert , Fox Robert O. , Horwich Arthur L.
Published: July 2002

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound cochaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen–deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel β-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the β-strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.
Submillisecond protein folding kinetics studied by ultrarapid mixing
Authors: Chan Chi-Kin , Hu Yi , Takahashi Satoshi , Rousseau Denis L. , Eaton William A. , Hofrichter James
Published: July 2002

An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome
c
folding kinetics in the previously inaccessible time range 80 μs to 3 ms. To eliminate the heme–ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The fluorescence decrease is biphasic. There is an unresolved process with τ < 50 μs, followed by a slower, exponential process with τ = 600 μs at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.