bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene
ERCC1
Authors: Sargent R. Geoffrey , Rolig Rhonda L. , Kilburn April E. , Adair Gerald M. , Wilson John H. , Nairn Rodney S.
Published: July 2002

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in
Saccharomyces cerevisiae
and
Drosophila melanogaster
, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene
ERCC1
, the hamster homologue to the
S. cerevisiae RAD10
gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (
APRT
) gene then were constructed at the endogenous
APRT
locus in
ERCC1
−
and
ERCC1
+
cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental
APRT
+
tandem duplication could give rise to
APRT
−
cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual
APRT
−
products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the
ERCC1
deficiency on gene stability, because
ERCC1
−
cell lines with a single wild-type copy of the
APRT
gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in
ERCC1
−
cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the
ERCC1
gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in
ERCC1
−
cells are repaired by illegitimate recombination.
An α-
E-catenin
gene trap mutation defines its function in preimplantation development
Authors: Torres Miguel , Stoykova Anastassia , Huber Otmar , Chowdhury Kamal , Bonaldo Paolo , Mansouri Ahmed , Butz Stefan , Kemler Rolf , Gruss Peter
Published: July 2002

Catenins are proteins associated with the cytoplasmic domain of cadherins, a family of transmembrane cell adhesion molecules. The cadherin–catenin adhesion system is involved in morphogenesis during development and in the maintenance of the integrity of different tissue types. Using a gene trap strategy, we have isolated a mouse mutation for the gene encoding the α-E-catenin. This form of the α-catenin appears frequently coexpressed with E-cadherin in epithelial cell types. The mutation obtained eliminates the carboxyl-terminal third of the protein but nevertheless provokes a complete loss-of-function phenotype. Homozygous mutants show disruption of the trophoblast epithelium (the first differentiated embryonic tissue), and development is consequently blocked at the blastocyst stage. This phenotype parallels the defects observed in E-cadherin mutant embryos. Our results show the requirement of the α-E-catenin carboxy terminus for its function and represent evidence of the role of the α-E-catenin
in vivo
, identifying this molecule as the natural partner of the E-cadherin in trophoblast epithelium.
Evolutionary mechanisms and population dynamics of the third variable envelope region of HIV within single hosts
Authors: Yamaguchi Yumi , Gojobori Takashi
Published: July 2002

Clonal diversifications of HIV virus were monitored by periodic samplings on each of the six patients with regard to 183- to 335-bp segments of the
env
gene, which invariably included the functionally critical V3 region. Subsequently, six individual phylogenetic trees of viral variants were constructed. It was found that at one time or another during the course of disease progression, viral variants were inexplicably released from a strong negative selection against nonsynonymous base substitutions, possibly indicating positive selection. This resulted in concentrated amino acid substitutions at five specific sites within the V3 region. It was noted that these sites were often involved as antigenic determinants that provoked the host immune response and that these sites were also involved in the determination of viral phenotypes as to their cell tropism, syncytium formation capability, and replication rates.
Transcriptional arrest:
Escherichia coli
RNA polymerase translocates backward, leaving the 3′ end of the RNA intact and extruded
Authors: Komissarova Natalia , Kashlev Mikhail
Published: July 2002

RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked. Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of
Escherichia coli
RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3′ end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme. The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage. Secondary structure elements or the 5′ end of the transcript can prevent the isomerization by blocking the RNA threading. The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation.
Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons
Authors: Jurka Jerzy
Published: July 2002

It is commonly accepted that the reverse-transcribed cellular RNA molecules, called retroposons, integrate at staggered breaks in mammalian chromosomes. However, unlike what was previously thought, most of the staggered breaks are not generated by random nicking. One of the two nicks involved is primarily associated with the 5′-TTAAAA hexanucleotide and its variants derived by a single base substitution, particularly A → G and T → C. It is probably generated in the antisense strand between the consensus bases 3′-AA and TTTT complementary to 5′-TTAAAA. The sense strand is nicked at variable distances from the TTAAAA consensus site toward the 3′ end, preferably within 15–16 base pairs. The base composition near the second nicking site is also nonrandom at positions preceding the nick. On the basis of the observed sequence patterns it is proposed that integration of mammalian retroposons is mediated by an enzyme with endonucleolytic activity. The best candidate for such enzyme may be the reverse transcriptase encoded by the L1 non-long-terminal-repeat retrotransposon, which contains a freshly reported domain homologous to the apurinic/apyrimidinic (AP) endonuclease family [Martin, F., Olivares, M., Lopez, M. C. & Alonso, C. (1996)
Trends Biochem. Sci.
21, 283–285; Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996)
Cell
87, 905–916] and shows nicking
in vitro
with preference for targets similar to 5′-TTAAAA/3′-AATTTT consensus sequence [Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996)
Cell
87, 905–916]. A model for integration of mammalian retroposons based on the presented data is discussed.
Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization
Authors: Morrison Sean J. , Wright Douglas E. , Weissman Irving L.
Published: July 2002

We isolated hematopoietic stem cells (HSC) from mice treated with cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). All mobilized multipotent progenitor activity was contained in two populations: Thy-1
lo
Sca-1
+
Lin
−
Mac-1
−
CD4
−
c-kit
+
long-term reconstituting progenitors and Thy-1
lo
Sca-1
+
Lin
−
Mac-1
lo
CD4
−
transiently reconstituting progenitors. CY/G-CSF treatment drove both long-term and transient multipotent progenitors into cycle, leading to a more than 12-fold expansion in the number of long-term self-renewing HSC prior to mobilization. After CY and 2 days of G-CSF treatment the number of bone marrow HSC began to decline and the number of blood and splenic HSC increased. HSC continued to proliferate in the bone marrow and spleen through 8 days of G-CSF treatment, but HSC released into the blood tended to be in G
0
/G
1
phase. Mobilized multipotent progenitors isolated from the spleen were less efficient than normal bone marrow multipotent progenitors in engrafting irradiated mice but did not differ in colony forming unit-spleen (CFU-S) activity or single cell
in vitro
assays of primitive progenitor activity. The data suggest that mobilized HSC isolated from the spleen are less efficient at homing to and engrafting the bone marrow of irradiated recipient mice.
The structure of mitogen-activated protein kinase p38 at 2.1-Å resolution
Authors: Wang Zhulun , Harkins Paul C. , Ulevitch Richard J. , Han Jiahuai , Cobb Melanie H. , Goldsmith Elizabeth J.
Published: July 2002

The structure of mitogen-activated protein (MAP) kinase p38 has been solved at 2.1-Å to an
R
factor of 21.0%, making p38 the second low activity MAP kinase solved to date. Although p38 is topologically similar to the MAP kinase ERK2, the phosphorylation Lip (a regulatory loop near the active site) adopts a different fold in p38. The peptide substrate binding site and the ATP binding site are also different from those of ERK2. The results explain why MAP kinases are specific for different activating enzymes, substrates, and inhibitors. A model presented for substrate and activator interactions has implications for the evolution of protein kinase cascades.
Neuronal nitric oxide synthase alternatively spliced forms: Prominent functional localizations in the brain
Authors: Eliasson Mikael J. L. , Blackshaw Seth , Schell Michael J. , Snyder Solomon H.
Published: July 2002

Neuronal nitric-oxide synthase (nNOS) is subject to alternative splicing. In mice with targeted deletions of exon 2 (nNOS
Δ/Δ
), two alternatively spliced forms, nNOSβ and γ, which lack exon 2, have been described. We have compared localizations of native nNOSα and nNOSβ and γ by
in situ
hybridization and immunohistochemistry in wild-type and nNOS
Δ/Δ
mice. To assess nNOS catalytic activity in intact animals we localized citrulline, which is formed stoichiometrically with NO, by immunohistochemistry. nNOSβ is prominent in several brain regions of wild-type animals and shows 2-to 3-fold up-regulation in the cortex and striatum of nNOS
Δ/Δ
animals. The persistence of much nNOS mRNA and protein, and distinct citrulline immunoreactivity (cit-IR) in the ventral cochlear nuclei and some cit-IR in the striatum and lateral tegmental nuclei, indicate that nNOSβ is a major functional form of the enzyme in these regions. Thus, nNOSβ, and possibly other uncharacterized splice forms, appear to be important physiological sources of NO in discrete brain regions and may account for the relatively modest level of impairment in nNOS
Δ/Δ
animals.
Trends in odor intensity for human and electronic noses: Relative roles of odorant vapor pressure vs. molecularly specific odorant binding
Authors: Doleman Brett J. , Severin Erik J. , Lewis Nathan S.
Published: July 2002

Response data were collected for a carbon black-polymer composite electronic nose array during exposure to homologous series of alkanes and alcohols. The mean response intensity of the electronic nose detectors and the response intensity of the most strongly driven set of electronic nose detectors were essentially constant for members of a chemically homologous odorant series when the concentration of each odorant in the gas phase was maintained at a constant fraction of the odorant’s vapor pressure. A similar trend is observed in human odor detection threshold values for these same homologous series of odorants. Because the thermodynamic activity of an odorant at equilibrium in a sorbent phase is equal to the partial pressure of the odorant in the gas phase divided by the vapor pressure of the odorant and because the activity coefficients are similar within these homologous series of odorants for sorption of the vapors into specific polymer films, the data imply that the trends in detector response can be understood based on the thermodynamic tendency to establish a relatively constant concentration of sorbed odorant into each of the polymeric films of the electronic nose at a constant fraction of the odorant’s vapor pressure. Similarly, the data are consistent with the hypothesis that the odor detection thresholds observed in human psychophysical experiments for the odorants studied herein are driven predominantly by the similarity in odorant concentrations sorbed into the olfactory epithelium at a constant fraction of the odorant’s vapor pressure.
A genetic record of population isolation in pocket gophers during Holocene climatic change
Authors: Hadly Elizabeth A. , Kohn Michael H. , Leonard Jennifer A. , Wayne Robert K.
Published: July 2002

A long-standing question in Quaternary paleontology is whether climate-induced, population-level phenotypic change is a result of large-scale migration or evolution in isolation. To directly measure genetic variation through time, ancient DNA and morphologic variation was measured over 2,400 years in a Holocene sequence of pocket gophers (
Thomomys talpoides
) from Lamar Cave, Yellowstone National Park, Wyoming. Ancient specimens and modern samples collected near Lamar Cave share mitochondrial cytochrome
b
sequences that are absent from adjacent localities, suggesting that the population was isolated for the entire period. In contrast, diastemal length, a morphologic character correlated with body size and nutritional level, changed predictably in response to climatic change. Our results demonstrate that small mammal populations can experience the long-term isolation assumed by many theoretical models of microevolutionary change.
Molecular characterization of a common fragile site (
FRA7H
) on human chromosome 7 by the cloning of a simian virus 40 integration site
Authors: Mishmar Dan , Rahat Ayelet , Scherer Stephen W. , Nyakatura Gerald , Hinzmann Bernd , Kohwi Yoshinori , Mandel-Gutfroind Yael , Lee Jeffrey R. , Drescher Bernd , Sas Dean E. , Margalit Hanah , Platzer Mattias , Weiss Aryeh , Tsui Lap-Chee , Rosenthal André , Kerem Batsheva
Published: July 2002

Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by fluorescent
in situ
hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7,
FRA7H
. A region of 161 kb spanning
FRA7H
was defined and sequenced. Several regions with a potential unusual DNA structure, including high-flexibility, low-stability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published
FRA3B
sequence and the putative partial
FRA7G
, which also revealed an impressive cluster of regions with high flexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility.
Mismatch repair deficiency associated with overexpression of the
MSH3
gene
Authors: Marra Giancarlo , Iaccarino Ingram , Lettieri Teresa , Roscilli Giuseppe , Delmastro Paola , Jiricny Josef
Published: July 2002

We tested the ability of recombinant hMutSα (hMSH2/hMSH6) and hMutSβ (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion–deletion loops was restored by hMutSα, only the latter substrates were addressed in extracts supplemented with hMutSβ. hMutSα was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (
DHFR
) locus, which also contains the
MSH3
gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSα and MutSβ. As a rule, MSH2 is primarily complexed with MSH6. MutSα is thus relatively abundant in mammalian cell extracts, whereas MutSβ levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSβ. This leads to degradation of the partnerless MSH6 and depletion of MutSα. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSβ. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond
et al.
[Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997)
Proc. Natl. Acad. Sci. USA
94, 10144–10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.
Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase
Authors: Endres Matthias , Laufs Ulrich , Huang Zhihong , Nakamura Tadashi , Huang Paul , Moskowitz Michael A. , Liao James K.
Published: July 2002

The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurological function in normocholesterolemic mice. The up-regulation of eNOS by HMG-CoA reductase inhibitors is not associated with changes in serum cholesterol levels, but is reversed by cotreatment withl-mevalonate and by the downstream isoprenoid, geranylgeranyl pyrophosphate and not by farnesyl pyrophosphate. The blood flow and neuroprotective effects of HMG-CoA reductase inhibitors are completely absent in eNOS-deficient mice, indicating that enhanced eNOS activity by HMG-CoA reductase inhibitors is the predominant if not the only mechanism by which these agents protect against cerebral injury. Our results suggest that HMG-CoA reductase inhibitors provide a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.
Successful search for a resistance gene in tomato targeted against a virulence factor of a fungal pathogen
Authors: Laugé Richard , Joosten Matthieu H. A. J. , Haanstra Jair P. W. , Goodwin Paul H. , Lindhout Pim , De Wit Pierre J. G. M.
Published: July 2002

The interaction between tomato and its fungal pathogenCladosporium fulvumcomplies with the gene-for-gene system, in which specific recognition of fungal proteins by plant genotypes with matching resistance genes results in host resistance. Two proteins, ECP1 and ECP2, secreted byC. fulvumduring infection, are required for full virulence of the fungus on tomato. We chose the most important virulence factor, ECP2, for a targeted search for hypersensitive response (HR)-based resistance among a collection of tomato genotypes. By screening with recombinant potato virus X that expresses theEcp2 gene, we identified four lines that respond with HR toward ECP2. The capacity to recognize ECP2 and induce HR is sufficient to confer resistance in tomato againstC. fulvumproducing ECP2. Resistance is based on a single dominant gene, which we have designatedCf-ECP2, for resistance toC. fulvumthrough recognition of ECP2. Accordingly, anEcp2-minus strain created by gene replacement is pathogenic onCf-ECP2plants. However, due to lack of ECP2 the mutant strain is only weakly virulent. All strains of a worldwide collection ofC. fulvumstrains that were tested were found to produce a HR-inducing ECP2 protein. Because theCf-ECP2gene operates through recognition of an important virulence factor, we expect it will confer durable resistance againstC. fulvum. A similar targeted approach should allow the discovery of new valuable resistance genes in other pathosystems.
cyclops
encodes a nodal-related factor involved in midline signaling
Authors: Rebagliati Michael R. , Toyama Reiko , Haffter Pascal , Dawid Igor B.
Published: July 2002

Ventral structures in the central nervous system are patterned by signals emanating from the underlying mesoderm as well as originating within the neuroectoderm. Mutations in the zebrafish,
Danio rerio
, are proving instrumental in dissecting these midline signals. The
cyclops
mutation leads to a loss of medial floor plate and to severe deficits in ventral forebrain development, leading to cyclopia. Here, we report that the
cyclops
locus encodes the nodal-related protein Ndr2, a member of the transforming growth factor type β superfamily of factors. The evidence includes identification of a missense mutation in the initiation codon and rescue of the
cyclops
phenotype by expression of
ndr2
RNA, here renamed “
cyclops
.” Thus, in interaction with other molecules implicated in these processes such as sonic hedgehog and one-eyed-pinhead, cyclops is required for ventral midline patterning of the embryonic central nervous system.
Oocytes are a source of catecholamines in the primate ovary: Evidence for a cell–cell regulatory loop
Authors: Mayerhofer Artur , Smith Gary D. , Danilchik Michael , Levine Jon E. , Wolf Don P. , Dissen Gregory A. , Ojeda Sergio R.
Published: July 2002

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine β-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes—but not those from which the oocytes had been removed—responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a β-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced β-adrenoreceptor activation.
FRAXA and FRAXE: Evidence against segregation distortion and for an effect of intermediate alleles on learning disability
Authors: Teague J. W. , Morton N. E. , Dennis N. R. , Curtis G. , McKechnie N. , Macpherson J. N. , Murray A. , Pound M. C. , Sharrock A. J. , Youings S. A. , Jacobs P. A.
Published: July 2002

There have been several claims of segregation distortion (meiotic drive) for loci associated with diseases caused by trinucleotide repeats, leading us to test for this phenomenon in a large study of the X-linked loci FRAXA and FRAXE. We found no evidence of meiotic drive in females and no convincing evidence in males, where the limitation of risk to daughters creates a testing bias for alleles of interest. Alleles for pre- and full mutation, intermediate alleles, and common alleles were analyzed separately, with the same negative results that are extended in the discussion to claims of meiotic drive for other diseases. On the other hand, an excess risk of learning difficulties was confirmed for intermediate FRAXA alleles (relative risk, 2.58 ± .74) and suggested for intermediate FRAXE alleles. The penetrance of learning difficulty is low, the risk being estimated as .039 for FRAXA common alleles and .101 for intermediate alleles. Because of their lower gene frequency, full mutations are a less frequent cause of learning difficulty than intermediate alleles, which contribute .0020 to total prevalence and .0012 to attributable prevalence of learning difficulty.
Strand asymmetry of CpG transitions as indicator of G
1
phase-dependent origin of multiple tumorigenic p53 mutations in stem cells
Authors: Rodin Sergei N. , Rodin Andrew S.
Published: July 2002

In dividing cells, expression of mutations is DNA strand symmetric. Of all mutations originating
de novo
in nondividing cells, only those in the transcribed (noncoding) strand are immediately expressed in mRNA and protein. In contrast, any new mutation in the nontranscribed (coding) strand remains unexpressed until the cells enter S phase and begin proliferation. This previously unrecognized difference enables us to examine the cell cycle-dependent origin of multiple tumorigenic mutations in stem cells. The human
p53
gene, which acts as a gatekeeper in the control of G
1
to S phase transition, was chosen for the analysis. Of all multiple mutations contained in p53 databases, we have tested in detail CpG transitions. Three features of CpG sites dictate this choice: C → T transitions at methylated
m
CpG are the direct product of
m
C deamination and are replication-independent; it is easy to identify the strand bearing a primary
m
C → T event because C → T on the transcribed strand appears as G → A on the nontranscribed strand; and CpG transitions are the most frequent (as both singular and multiple occurrences) tumor-related p53 mutations. The origin of double nonsilent CpG transitions in nondividing cells predicts a significant excess of the heterostrand (C → T, G → A) doublets over the homostrand (C → T, C → T and G → A, G → A) doublets. For
p53
, we found such an excess. Based on this result, along with the results of three other tests reported here, we conclude that the majority of multiple p53 mutations from human tumors occurred in quiescent stem cells.
Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism
Authors: Wen Yingxia , Yue Zhenyu , Shatkin Aaron J.
Published: July 2002

Mammalian capping enzymes are bifunctional proteins with both RNA 5′-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5′-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO
4
3−
and independent of Mg
2+
, providing additional evidence for phosphate removal from RNA 5′ ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211–597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. RNA binding was increased by GTP, and a guanylylation-defective, active-site mutant was not affected. Ala substitution at positions required for the formation of the enzyme-GMP capping intermediate (R315, R530, K533, or N537) also eliminated poly (U) binding, while proteins with conservative substitutions at these sites retained binding but not guanylyltransferase activity. These results demonstrate that the guanylyltransferase domain of mammalian capping enzyme specifies nuclear localization and RNA binding. Association of capping enzyme with nascent transcripts may act in synergy with RNA polymerase II binding to ensure 5′ cap formation.
Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in
Mycobacterium tuberculosis
Authors: Alland David , Kramnik Igor , Weisbrod Torin R. , Otsubo Lisa , Cerny Rosaria , Miller Lincoln P. , Jacobs William R. , Bloom Barry R.
Published: July 2002

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of
Mycobacterium tuberculosis
, latency. Because of the absence of poly(A)
+
mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the
in vitro
effect of the antibiotic isoniazid on
M. tuberculosis
, and three previously uncharacterized isoniazid-induced genes,
iniA
,
iniB
, and
iniC
, were identified. The
iniB
gene has homology to cell wall proteins, and
iniA
contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The
iniA
gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.
SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (
COL
7A1
) promoter by transforming growth factor β
Authors: Vindevoghel Laurence , Lechleider Robert J. , Kon Atsushi , de Caestecker Mark P. , Uitto Jouni , Roberts Anita B. , Mauviel Alain
Published: July 2002

The human type VII collagen gene (
COL7A1
) recently has been identified as an immediate-early response gene for transforming growth factor β (TGF-β)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4−/− breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-β response element in the region −496/−444 of the
COL7A1
promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1–5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-β-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione
S
-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the
COL7A1
SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of
COL7A1
SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.
Aggregation of the human high affinity immunoglobulin G receptor (FcγRI) activates both tyrosine kinase and G protein-coupled phosphoinositide 3-kinase isoforms
Authors: Melendez Alirio J. , Gillooly David J. , Harnett Margaret M. , Allen Janet M.
Published: July 2002

Phosphoinositide 3-kinases (PI3-kinases) play an important role in the generation of lipid second messengers and the transduction of a myriad of biological responses. Distinct isoforms have been shown to be exclusively activated either by tyrosine kinase-coupled or G protein-coupled receptors. We show here, however, that certain nonclassical receptors can couple to both tyrosine kinase- and G protein-dependent isoforms of PI3-kinase: thus, aggregation of FcγRI, the human high affinity IgG receptor, on monocytes unusually leads to activation of both of these types of PI3-kinase. After aggregation of FcγRI, phosphatidylinositol 3,4,5-triphosphate (PIP3) levels rise rapidly in interferon γ-primed cells, reaching a peak within 30 sec. Moreover, and in contrast to the situation observed after stimulation of these cells with either insulin or ATP, which exclusively activate the tyrosine kinase- and G protein-coupled forms of PI3-kinase, respectively, PIP3levels remain elevated up to 15 min after receptor aggregation. We show here that although the initial peak results from transient activation of the p85-dependent p110 isoform of PI-3kinase, presumably through recruitment of tyrosine kinases by the γ chain, the later sustained rise of PIP3results from activation of the G protein βγ subunit-sensitive isoform, p110γ. This finding indicates that receptors lacking an intrinsic signaling motif, such as FcγRI, can recruit both tyrosine kinase and G protein-coupled intracellular signaling molecules and thereby initiate cellular responses.
Expression of Q227L-Gα
s
in MCF-7 human breast cancer cells inhibits tumorigenesis
Authors: Chen Jianghao , Bander Jeffry A. , Santore Tara Ann , Chen Yibang , Ram Prahlad T. , Smit Martine J. , Iyengar Ravi
Published: July 2002

The effects of expression of mutant (Q227L)-activated Gα
s
and elevation of cAMP on mitogen-activating protein kinase (MAPK) activity and the transformed phenotype were studied in the MCF-7 human mammary epithelial cell line. Elevation of cAMP partially inhibited the epidermal growth factor-stimulated DNA synthesis and the intrinsic MAPK (ERK-1 and ERK-2) of serum-starved MCF-7 cells. Addition of 8Br-cAMP or expression of mutant (Q227L)-activated Gα
s
in MCF-7 cells blocked the ability of these cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. 8Br-cAMP in the culture medium also blocked estrogen stimulation of MCF-7 cell proliferation
in vitro
. MCF-7 cells expressing Q227L-Gα
s
grew very slowly
in vitro
, and when these cells were injected s.c. into athymic mice implanted with estrogen pellets, the frequency of tumor formation was reduced greatly and the sizes of the tumors formed were much smaller than those in mice injected with MCF-7 cells that had been transfected with the empty vector. These results indicate that the intracellular levels of cAMP in transformed mammary epithelial cells can be a crucial factor in determining the expression of the transformed phenotype. Interactions between the G
s
/adenylyl cyclase and MAPK-1,2 signaling pathways could be one mechanism by which expression of the transformed phenotype in mammary epithelial cells are regulated.
Seeking an ancient enzyme in
Methanococcus jannaschii
using
orf
, a program based on predicted secondary structure comparisons
Authors: Aurora Rajeev , Rose George D.
Published: July 2002

We have developed a simple procedure to identify protein homologs in genomic databases. The program, called
orf
, is based on comparisons of predicted secondary structure. Protein structure is far better conserved than amino acid sequence, and structure-based methods have been effective in exploiting this fact to find homologs, even among proteins with scant sequence identity.
orf
is a secondary structure-based method that operates solely on predictions from sequence and requires no experimentally determined information about the structure. The approach is illustrated by an example: Thymidylate synthase, a highly conserved enzyme essential to thymidine biosynthesis in both prokaryotes and eukaryotes, is thought to be used by
Archaea
, but a corresponding gene has yet to be identified. Here, a candidate thymidylate synthase is identified as a previously unassigned open reading frame from the genome of
Methanococcus jannaschii, viz.,
MJ0757. Using primary structure information alone, the optimally aligned sequence identity between MJ0757 and
Escherichia coli
thymidylate synthase is 7%, well below the threshold of sensitivity for detection by sequence-based methods.
Biochemical evidence for pathogenicity of rhodopsin kinase mutations correlated with the Oguchi form of congenital stationary night blindness
Authors: Khani Shahrokh C. , Nielsen Lori , Vogt Todd M.
Published: July 2002

Rhodopsin kinase (RK), a rod photoreceptor cytosolic enzyme, plays a key role in the normal deactivation and recovery of the photoreceptor after exposure to light. To date, three different mutations in the RK locus have been associated with Oguchi disease, an autosomal recessive form of stationary night blindness in man characterized in part by delayed photoreceptor recovery [Yamamoto, S., Sippel, K. C., Berson, E. L. & Dryja, T. P. (1997)
Nat. Genet.
15, 175–178]. Two of the mutations involve exon 5, and the remaining mutation occurs in exon 7. Known exon 5 mutations include the deletion of the entire exon sequence [HRK
(X5 del)
] and a missense change leading to a Val380Asp substitution in the encoded product (HRK
V380D
). The mutation in exon 7 is a 4-bp deletion in codon 536 leading to premature termination of the encoded polypeptide [HRK
S536(4-bp del)
]. To provide biochemical evidence for pathogenicity of these mutations, wild-type human rhodopsin kinase (HRK) and mutant forms HRK
V380D
and HRK
S536(4-bp del)
were expressed in COS7 cells and their activities were compared. Wild-type HRK catalyzed light-dependent phosphorylation of rhodopsin efficiently. In contrast, both mutant proteins were markedly deficient in catalytic activity with HRK
V380D
showing virtually no detectible activity and HRK
S536(4-bp del)
only minimal light-dependent activity. These results provide biochemical evidence to support the pathogenicity of the RK mutations in man.
In vitro
disassembly and reassembly of an ABC transporter, the histidine permease
Authors: Liu Pei-Qi , Ames Giovanna Ferro-Luzzi
Published: July 2002

The membrane-bound complex of the
Salmonella typhimurium
periplasmic histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP. The complex hydrolyzes ATP upon induction of the activity by the liganded soluble receptor, the periplasmic histidine-binding protein, HisJ. Here we take advantage of the modular organization of this system to show that the nucleotide-binding component can be stripped off the integral membrane components, HisQ and HisM. The complex can be reconstituted by using the HisP-depleted membranes containing HisQ and HisM and pure soluble HisP. We show that HisP has high affinity for the HisP-depleted complex, HisQM, and that two HisP molecules are recruited independently of each other for each HisQM unit. The
in vitro
reassembled complex has entirely normal properties, responding to HisJ and ATPase inhibitors with the same characteristics as the original complex and in contrast to those of soluble HisP. These results show that HisP is absolutely required for ATP hydrolysis, that HisQM cannot hydrolyze ATP, that HisP depends on HisQM to relay the inducing signal from the soluble receptor, HisJ, and that HisQM regulates the ATPase activity of HisP. We also show that HisP changes conformation upon exposure to phospholipids.
Stable transformation of the yellow fever mosquito,
Aedes aegypti
, with the
Hermes
element from the housefly
Authors: Jasinskiene Nijole , Coates Craig J. , Benedict Mark Q. , Cornel Anthony J. , Rafferty Cristina Salazar , James Anthony A. , Collins Frank H.
Published: July 2002

The mosquito
Aedes aegypti
is the world’s most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild
Ae. aegypti
populations to support virus replication. The germ-line transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified
Hermes
transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the
Drosophila melanogaster cinnabar (cn)
gene was used to transform a white-eyed recipient strain of
Ae. aegypti
. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G
0
adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the
cn
+
-carrying transposon. The
cn
+
gene functioned as a semidominant transgene and segregated in Mendelian ratios.
Hermes
shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.
Optimal prediction of underresolved dynamics
Authors: Chorin Alexandre J. , Kast Anton P. , Kupferman Raz
Published: July 2002

A method is presented for computing the average solution of
problems that are too complicated for adequate resolution, but where
information about the statistics of the solution is available. The
method involves computing average derivatives by interpolation based on
linear regression, and an updating of a measure constrained by the
available crude information. Examples are given.
Characterization of functionally active subribosomal particles from
Thermus aquaticus
Authors: Khaitovich Philipp , Mankin Alexander S. , Green Rachel , Lancaster Laura , Noller Harry F.
Published: July 2002

Peptidyl transferase activity of
Thermus aquaticus
ribosomes is resistant to the removal of a significant number of ribosomal proteins by protease digestion, SDS, and phenol extraction. To define the upper limit for the number of macromolecular components required for peptidyl transferase, particles obtained by extraction of
T. aquaticus
large ribosomal subunits were isolated and their RNA and protein composition was characterized. Active subribosomal particles contained both 23S and 5S rRNA associated with notable amounts of eight ribosomal proteins. N-terminal sequencing of the proteins identified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active in peptide bond formation, was not found. These findings, together with the results of previous reconstitution experiments, reduce the number of possible essential macromolecular components of the peptidyl transferase center to 23S rRNA and ribosomal proteins L2 and L3. Complete removal of ribosomal proteins from
T. aquaticus
rRNA resulted in loss of tertiary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase hydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resistance to SDS extraction than 30S subunits are compatible with a proposed structural organization of the 50S subunit involving an RNA “cage” surrounding a core of a subset of ribosomal proteins.
Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays
Authors: Gao Changshou , Mao Shenlan , Lo Chih-Hung L. , Wirsching Peter , Lerner Richard A. , Janda Kim D.
Published: July 2002

The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein. We developed a phagemid format wherein antibody heavy- and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively. Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface. Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays. Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities.
“Global” cell replacement is feasible via neural stem cell transplantation: Evidence from the dysmyelinated
shiverer
mouse brain
Authors: Yandava Booma D. , Billinghurst Lori L. , Snyder Evan Y.
Published: July 2002

Many diseases of the central nervous system (CNS), particularly those of genetic, metabolic, or infectious/inflammatory etiology, are characterized by “global” neural degeneration or dysfunction. Therapy might require widespread neural cell replacement, a challenge not regarded conventionally as amenable to neural transplantation. Mouse mutants characterized by CNS-wide white matter disease provide ideal models for testing the hypothesis that neural stem cell transplantation might compensate for defective neural cell types in neuropathologies requiring cell replacement throughout the brain. The oligodendrocytes of the dysmyelinated
shiverer
(
shi
) mouse are “globally” dysfunctional because they lack myelin basic protein (MBP) essential for effective myelination. Therapy, therefore, requires widespread replacement with MBP-expressing oligodendrocytes. Clonal neural stem cells transplanted at birth—using a simple intracerebroventricular implantation technique—resulted in widespread engraftment throughout the
shi
brain with repletion of MBP. Accordingly, of the many donor cells that differentiated into oligodendroglia—there appeared to be a shift in the fate of these multipotent cells toward an oligodendroglial fate—a subgroup myelinated up to 52% (mean = ≈40%) of host neuronal processes with better compacted myelin of a thickness and periodicity more closely approximating normal. A number of recipient animals evinced decrement in their symptomatic tremor. Therefore, “global” neural cell replacement seems feasible for some CNS pathologies if cells with stem-like features are used.
mRNA cap recognition: Dominant role of enhanced stacking interactions between methylated bases and protein aromatic side chains
Authors: Hu Guanghui , Gershon Paul D. , Hodel Alec E. , Quiocho Florante A.
Published: July 2002

We have determined, by high resolution x-ray analysis, 10 structures comprising the mRNA cap-specific methyltransferase VP39 or specific mutants thereof in the presence of methylated nucleobase analogs (N1-methyladenine, N3-methyladenine, N1-methylcytosine, N3-methylcytosine) and their unmethylated counterparts, or nucleoside N7-methylguanosine. Together with solution affinity studies and previous crystallographic data for N7-methylguanosine and its phosphorylated derivatives, these data demonstrate that only methylated, positively charged bases are bound, indicating that their enhanced stacking with two aromatic side chains of VP39 (Tyr 22 and Phe 180) plays a dominant role in cap recognition. Four key features characterize this stacking interaction: (
i
) near perfect parallel alignment between the sandwiched methylated bases and aromatic side chains, (
ii
) substantial areas of overlap in the two-stacked rings, (
iii
) a 3.4-Å interplanar spacing within the overlapping region, and (
iv
) positive charge in the heterocyclic nucleobase.