bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional association between Arf and RalA in active phospholipase D complex
Authors: Luo Jing-Qing , Liu Xin , Frankel Paul , Rotunda Thuy , Ramos Miguel , Flom Judith , Jiang Hong , Feig Larry A. , Morris Andrew J. , Kahn Richard A. , Foster David A.
Published: July 2002

Activation of phospholipase D1 (PLD1) by Arf has been implicated in vesicle transport and membrane trafficking. PLD1 has also been shown to be associated with the small GTPase RalA, which functions downstream from Ras in a Ras–RalA GTPase cascade that facilitates intracellular signal transduction. Although PLD1 associates directly with RalA, RalA has no effect upon the activity of PLD1. However, PLD1 precipitated from cell lysates with immobilized glutathione
S
-transferase–RalA fusion protein is active. This suggests the presence of an additional activating factor in the active RalA–PLD1 complexes. Because Arf stimulates PLD1, we looked for the presence of Arf in the active RalA–PLD1 complexes isolated from v-Src- and v-Ras-transformed cell lysates. Low levels of Arf protein were detected in RalA–PLD1 complexes; however, if guanosine 5′-[γ-thio]triphosphate was added to activate Arf and stimulate translocation to the membrane, high levels of Arf were precipitated by RalA from cell lysates. Interestingly, deletion of 11 amino-terminal amino acids unique to Ral GTPases, which abolished the ability of RalA to precipitate PLD activity, prevented the association between RalA and Arf. Brefeldin A, which inhibits Arf GDP–GTP exchange, inhibited PLD activity in v-Src- and v-Ras-transformed cells but not in the nontransformed cells, suggesting that the association of Arf with RalA is required for the increased PLD activity induced by v-Src and v-Ras. These data implicate Arf in the transduction of intracellular signals activated by v-Src and mediated by the Ras–RalA GTPase cascade. Because both Arf and PLD1 stimulate vesicle formation in the Golgi, these data raise the possibility that vesicle formation and trafficking may play a role in the transduction of intracellular signals.
Translation of cytochrome
f
is autoregulated through the 5′ untranslated region of
petA
mRNA in
Chlamydomonas
chloroplasts
Authors: Choquet Y. , Stern D. B. , Wostrikoff K. , Kuras R. , Girard-Bascou J. , Wollman F.-A.
Published: July 2002

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in
Chlamydomonas reinhardtii
: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome
b
6
f
complex and show that negative autoregulation of cytochrome
f
translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5′ untranslated region of
petA
mRNA, which encodes cytochrome
f
, and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome
f
translation explains both the decreased rate of cytochrome
f
synthesis
in vivo
in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the
atpA
5′ untranslated region, cytochrome
f
no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric
petA
-
aadA
-
rbcL
gene was shown to depend on the state of assembly of cytochrome
b
6
f
complexes and on the accumulation of the C-terminal domain of cytochrome
f
. We discuss the possible ubiquity of the CES process in organellar protein biogenesis.
Selective killing of preneoplastic and neoplastic cells by methotrexate with leucovorin
Authors: Chow Ming , Rubin Harry
Published: July 2002

Three sublines of NIH 3T3 cells had the properties of non-neoplastic, preneoplastic, and neoplastic cells, respectively. The closer the cells were to neoplastic behavior, characterized by continuing growth at high density, the slower they multiplied at lower density. Under the conditions of high population density and low calf serum concentration used in the assay for transformed focus formation, the transformed or neoplastic cells were much more sensitive to killing by methotrexate (MTX) than were non-neoplastic cells in the same culture. This differential sensitivity of neoplastic cells was far more pronounced in molecular, cellular, and developmental biology medium 402 (MCDB 402) than in DMEM. It is associated with the presence in MCDB 402 of folinic acid, known clinically as leucovorin, which is a reduced form of the folic acid present in DMEM. Although leucovorin had been shown to selectively spare normal bone marrow and intestine in animals from the killing effect of MTX on tumor cells, we demonstrate the preferential killing of neoplastic over non-neoplastic cells of the same derivation. Neither neoplastic nor non-neoplastic cells were killed once they had stopped multiplying at their respective saturation densities. The development of the light foci characteristic of the preneoplastic cells was less sensitive to MTX than the formation of the dense foci produced by the fully neoplastic cells. The system should serve as a valuable model to establish basic principles and optimal conditions for selective killing of neoplastic cells by chemotherapeutic drugs.
The sequences of small proteins are not extensively optimized for rapid folding by natural selection
Authors: Kim David E. , Gu Hongdi , Baker David
Published: July 2002

The thermodynamic stabilities of small protein domains are clearly subject to natural selection, but it is less clear whether the rapid folding rates typically observed for such proteins are consequences of direct evolutionary optimization or reflect intrinsic physical properties of the polypeptide chain. This issue can be investigated by comparing the folding rates of laboratory-generated protein sequences to those of naturally occurring sequences provided that the method by which the sequences are generated has no kinetic bias. Herein we report the folding thermodynamics and kinetics of 12 heavily mutated variants of the small IgG binding domain of protein L retrieved from high-complexity combinatorial libraries by using a phage-display selection for proper folding that does not discriminate between rapidly and slowly folding proteins. Although the stabilities of all variants were decreased, many of the variants fold faster than wild type. Taken together with similar results for the src homology 3 domain, this observation suggests that the sequences of small proteins have not been extensively optimized for rapid folding; instead, rapid folding appears to be a consequence of selection for stability.
Osmotic pressure contribution of albumin to colloidal interactions
Authors: Singh-Zocchi Mukta , Andreasen Anita , Zocchi Giovanni
Published: July 2002

Two surfaces that come in close contact in a solution with macromolecules present experience an attractive force caused by the osmotic pressure. We present a measurement of this effect by using a micrometer-sized sphere bound to a flat plate through a single molecular attachment in an albumin-containing solution. We obtain the osmotic part of the interaction potential with a resolution of <1 nm and a fraction of
kT
room
. This attractive interaction is seen to have a range comparable to the size of the albumin molecule. The results are broadly in agreement with a geometric model first proposed by Asakura and Oosawa.
Species richness and resource availability: A phylogenetic analysis of insects associated with trees
Authors: Kelly C. K. , Southwood T. R. E.
Published: July 2002

The data on the number of species of insects associated with various trees in Britain have been reanalyzed to factor out possible bias from phylogenetic effects. It was found that tree availability (range and abundance) continues to provide a good predictor (r= 0.852) of insect-species richness, slightly better than straightforward cross-species analyses. Of the two components of tree availability, tree abundance gives a much better prediction than tree range. The species richness on trees of major taxa with similar trophic habits (Lepidoptera and Hymenoptera/Symphyta and the two suborders of the Homoptera—Auchenorrhyncha and Sternorrhyncha) shows positive correlations; there is thus no evidence of competitive exclusion at this taxonomic level.
A single nuclear transcript encoding mitochondrial RPS14 and SDHB of rice is processed by alternative splicing: Common use of the same mitochondrial targeting signal for different proteins
Authors: Kubo Nakao , Harada Kyuya , Hirai Atsushi , Kadowaki Koh-ichi
Published: July 2002

The rice mitochondrial genome has a sequence homologous to the gene for ribosomal protein S14 (
rps14
), but the coding sequence is interrupted by internal stop codons. A functional
rps14
gene was isolated from the rice nuclear genome, suggesting a gene-transfer event from the mitochondrion to the nucleus. The nuclear
rps14
gene encodes a long N-terminal extension showing significant similarity to a part of mitochondrial succinate dehydrogenase subunit B (SDHB) protein from human and a malarial parasite (
Plasmodium falciparum
). Isolation of a functional rice
sdhB
cDNA and subsequent sequence comparison to the nuclear
rps14
indicate that the 5′ portions of the two cDNAs are identical. The
sdhB
genomic sequence shows that the SDHB-coding region is divided into two exons. Surprisingly, the RPS14-coding region is located between the two exons. DNA gel blot analysis indicates that both
sdhB
and
rps14
are present at a single locus in the rice nucleus. These findings strongly suggest that the two gene transcripts result from a single mRNA precursor by alternative splicing. Protein blot analysis shows that the size of the mature RPS14 is 16.5 kDa, suggesting removal of the N-terminal 22.6-kDa peptide region. Considering that the rice mitochondrial genome lacks the
sdhB
gene but contains the
rps14
-related sequence, transfer of the
sdhB
gene seems to have occurred before the transfer of the
rps14
gene. The migration of the mitochondrial
rps14
sequence into the already existing
sdhB
gene could bestow the capacity for nuclear expression and mitochondrial targeting.
Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor
Authors: Koulen Peter , Kuhn Rainer , Wässle Heinz , Brandstätter Johann Helmut
Published: July 2002

Fast excitatory neurotransmission in the central nervous system is mediated through glutamate acting on ionotropic glutamate receptors. However, glutamate acting on metabotropic glutamate receptors (mGluRs) can also exert an inhibitory action. Here, we report by immunocytochemistry and physiology, to our knowledge, the first glutamate receptor to be found in terminals of photoreceptors in the mammalian retina—the group III metabotropic glutamate receptor mGluR8. Glutamate is the transmitter of photoreceptors, and thus mGluR8 functions as an autoreceptor. Activation of mGluR8 by the group III mGluR agonists
l
-2-amino-4-phosphonobutyrate and
l
-serine-
O
-phosphate, or by glutamate itself, evokes a decrease in the intracellular calcium ion concentration ([Ca
2+
]
i
) in isolated photoreceptors. This effect is blocked by the group III mGluR antagonists (RS)-α-methyl-4-phosphonophenylglycine and (RS)-α-methylserine-
O
-phosphate. Agonists for other classes of glutamate receptors—
n
-methyl-
d
-aspartic acid, quisqualic acid, kainic acid, or (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid—have no effect on the [Ca
2+
]
i
in isolated photoreceptors. The down-regulation of the [Ca
2+
]
i
in photoreceptors by mGluR8 provides evidence for an inhibitory feedback loop at the photoreceptor synapse in the mammalian retina. This negative feedback may be a mechanism for the fine adjustment of the light-regulated release of glutamate from photoreceptors and may serve as a safety device against excitotoxic levels of release at this tonic synapse. Such a mechanism may provide a model for feedback inhibition in other parts of the central nervous system.
The complete chloroplast DNA sequence of the green alga
Nephroselmis olivacea
: Insights into the architecture of ancestral chloroplast genomes
Authors: Turmel Monique , Otis Christian , Lemieux Claude
Published: July 2002

Green plants seem to form two sister lineages: Chlorophyta, comprising the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae, and Streptophyta, comprising the Charophyceae and land plants. We have determined the complete chloroplast DNA (cpDNA) sequence (200,799 bp) of
Nephroselmis olivacea
, a member of the class (Prasinophyceae) thought to include descendants of the earliest-diverging green algae. The 127 genes identified in this genome represent the largest gene repertoire among the green algal and land plant cpDNAs completely sequenced to date. Of the
Nephroselmis
genes, 2 (
ycf81
and
ftsI
, a gene involved in peptidoglycan synthesis) have not been identified in any previously investigated cpDNA; 5 genes [
ftsW
,
rnE
,
ycf62
,
rnpB
, and
trnS(cga)
] have been found only in cpDNAs of nongreen algae; and 10 others (
ndh
genes) have been described only in land plant cpDNAs.
Nephroselmis
and land plant cpDNAs share the same quadripartite structure—which is characterized by the presence of a large rRNA-encoding inverted repeat and two unequal single-copy regions—and very similar sets of genes in corresponding genomic regions. Given that our phylogenetic analyses place
Nephroselmis
within the Chlorophyta, these structural characteristics were most likely present in the cpDNA of the common ancestor of chlorophytes and streptophytes. Comparative analyses of chloroplast genomes indicate that the typical quadripartite architecture and gene-partitioning pattern of land plant cpDNAs are ancient features that may have been derived from the genome of the cyanobacterial progenitor of chloroplasts. Our phylogenetic data also offer insight into the chlorophyte ancestor of euglenophyte chloroplasts.
Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage
Authors: Verdi Joseph M. , Bashirullah Arash , Goldhawk Donna E. , Kubu Chris J. , Jamali Mina , Meakin Susan O. , Lipshitz Howard D.
Published: July 2002

Neuronal cell fate decisions are directed in
Drosophila
by NUMB, a signaling adapter protein with two protein–protein interaction domains: a phosphotyrosine-binding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. Here we show that there are at least four human NUMB isoforms and that these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human NUMB protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the NOTCH receptor during mammalian neurogenesis.
The lack of a functional
p21
WAF1/CIP1
gene ameliorates progression to chronic renal failure
Authors: Megyesi Judit , Price Peter M. , Tamayo Esther , Safirstein Robert L.
Published: July 2002

Partial renal ablation leads to progressive renal insufficiency and is a model of chronic renal failure from diverse causes. We find that mice develop functional and morphologic characteristics of chronic renal failure after partial renal ablation, including glomerular sclerosis, systemic hypertension, and reduced glomerular filtration. However, we now report that littermates with a homozygous deletion of the gene for the cyclin-dependent kinase inhibitor,
p21
WAF1/CIP1
, do not develop chronic renal failure after ablation. The markedly different reactions of the
p21
(
+
/
+
) and
p21
(−/−) animals was not because of differences in glomerular number or degree of renal growth but rather because of the presence or absence of a normal
p21
gene. Although the reaction to the stress of renal ablation is both hyperplastic and hypertrophic in the presence of a functional
p21
gene, it would appear that the absence of the
p21
gene may induce a more hyperplastic reaction because proliferating-cell nuclear antigen expression, a marker of cell-cycle progression, in the renal epithelium of the remnant kidney was more than five times greater in the
p21
(−/−) mice than in the
p21
(
+
/
+
) animals. Because p21 is a potent inhibitor of the cell cycle, we speculate that p21 regulates the balance between hyperplasia and hypertrophy after renal ablation. We propose that this change in response inhibits the development of chronic renal failure. These studies suggest that controlling p21 function may ameliorate or even prevent progressive end-stage renal disease.
The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: Identification of a cyanobacterial homolog
Authors: Reumann Sigrun , Davila-Aponte Jennifer , Keegstra Kenneth
Published: July 2002

The known envelope membrane proteins of the chloroplastic protein import apparatus lack sequence similarity to proteins of other eukaryotic or prokaryotic protein transport systems. However, we detected a putative homolog of the gene encoding Toc75, the protein-translocating channel from the outer envelope membrane of pea chloroplasts, in the genome of the cyanobacterium
Synechocystis
sp. PCC 6803. We investigated whether the low sequence identity of 21% reflects a structural and functional relationship between the two proteins. We provide evidence that the cyanobacterial protein is also localized in the outer membrane. From this information and the similarity of the predicted secondary structures, we conclude that Toc75 and the cyanobacterial protein, referred to as SynToc75, are structural homologs.
synToc75
is essential, as homozygous null mutants were not recovered after directed mutagenesis. Sequence analysis indicates that SynToc75 belongs to a family of outer membrane proteins from Gram-negative bacteria whose function is not yet known. However, we demonstrate that these proteins are related to a specific group of prokaryotic secretion channels that transfer virulence factors, such as hemolysins and adhesins, across the outer membrane.
Inhibition of gene expression in human cells through small molecule-RNA interactions
Authors: Hwang Seongwoo , Tamilarasu Natarajan , Ryan Kevin , Huq Ikramul , Richter Sara , Still W. Clark , Rana Tariq M.
Published: July 2002

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′ end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from
d
-and
l
-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA.
In vitro
binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC
50
of ≈50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.
Positional enhancer-blocking activity of the chicken β-globin insulator in transiently transfected cells
Authors: Recillas-Targa Félix , Bell Adam C. , Felsenfeld Gary
Published: July 2002

It is thought that insulators demarcate transcriptionally and structurally independent chromatin domains. Insulators are detected by their ability to block enhancer–promoter interactions in a directional manner, and protect a transgene from position effects. Most studies are performed in stably transformed cells or organisms. Here we analyze the enhancer-blocking activity of the chicken β-globin insulator in transient transfection experiments in both erythroid and nonerythroid cell lines. We show that four tandem copies of a 90-bp fragment of this insulator were able to block an enhancer in these experiments. In circular plasmids, placement on either side of the enhancer reduced activity, but when the plasmid was linearized, the enhancer-blocking activity was observed only when the insulator was placed between the promoter and the enhancer. These observations are consistent with the position-dependent enhancer-blocking activity of the insulator observed in stable transformation experiments.
Mammalian
Trithorax
and
Polycomb
-group homologues are antagonistic regulators of homeotic development
Authors: Hanson Robin D. , Hess Jay L. , Yu Benjamin D. , Ernst Patricia , van Lohuizen Maarten , Berns Anton , van der Lugt Nathalie M. T. , Shashikant Cooduvalli S. , Ruddle Frank H. , Seto Masao , Korsmeyer Stanley J.
Published: July 2002

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (
Hox
) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in
Drosophila
maintain
Hox
expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of
Trx
.
Bmi-1
was first identified as a collaborator in
c-myc
-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member
Posterior sex combs
. Here, we note the axial-skeletal transformations and altered
Hox
expression patterns of
Mll
-deficient and
Bmi-1
-deficient mice were normalized when both
Mll
and
Bmi-1
were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from
Mll
-deficient compared with
Bmi-1
-deficient mice demonstrate reciprocal regulation of
Hox
genes as well as an integrated
Hoxc8
-lacZ reporter construct. Reexpression of
MLL
was able to overcome repression, rescuing expression of
Hoxc8
-lacZ in
Mll
-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although
Drosophila
Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target
Hox
gene.
α-Helix nucleation by a calcium-binding peptide loop
Authors: Siedlecka Monika , Goch Grażyna , Ejchart Andrzej , Sticht Heinrich , Bierzyǹski Andrzej
Published: July 2002

A 12-residue peptide AcDKDGDGYISAAENH
2
analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K = 10
5
M
−1
). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the α-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH
2
presented in this paper show that residues A13-Q16 form an α-helix of very high stability when the La
3+
ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1°C and 70% at 25°C. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters,
s
, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger
s
values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent α-helices is discussed. The problem of CD spectra of very short α-helices is also addressed.
The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments
Authors: Edskes Herman K. , Gray Vaughn T. , Wickner Reed B.
Published: July 2002

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a
regulator of nitrogen catabolism in
Saccharomyces
cerevisiae
. Ure2p
1–65
is the prion domain of Ure2p,
sufficient to propagate [URE3]
in vivo
.
We
show that full length Ure2p–green fluorescent protein (GFP) or a
Ure2p
1–65
-GFP fusion protein is aggregated in cells
carrying [URE3] but is evenly distributed in cells lacking the
[URE3] prion. This indicates that [URE3] involves a
self-propagating aggregation of Ure2p. Overexpression of
Ure2p
1–65
induces the
de novo
appearance of
[URE3] by 1,000-fold in a strain initially [ure-o], but cures
[URE3] from a strain initially carrying the [URE3] prion.
Overexpression of several other fragments of Ure2p or Ure2-GFP fusion
proteins also efficiently cures the prion. We suggest that
incorporation of fragments or fusion proteins into a putative [URE3]
“crystal” of Ure2p poisons its propagation.
On the mechanism by which vascular endothelial cells regulate their oxygen consumption
Authors: Clementi Emilio , Brown Guy Charles , Foxwell Neale , Moncada Salvador
Published: July 2002

Two enzymes, soluble guanylyl cyclase and cytochrome
c
oxidase, have been shown to be exquisitely sensitive to nitric
oxide (NO) at low physiological concentrations. Activation of the
soluble guanylyl cyclase by endogenous NO and the
consequent increase in the second messenger cyclic GMP are now known to
control a variety of biological functions. Cytochrome
c
oxidase, the terminal enzyme of the mitochondrial respiratory chain, is
inhibited by NO. However, it is not clear whether NO produced by the
constitutive NO synthase interacts with cytochrome
c
oxidase, nor is it known what the biological consequences of such an
interaction might be. We now show that NO generated by vascular
endothelial cells under basal and stimulated conditions modulates the
respiration of these cells in response to acute changes in oxygen
concentration. This action occurs at the cytochrome
c
oxidase and depends on influx of calcium. Thus, NO plays a
physiological role in adjusting the capacity of this enzyme to use
oxygen, allowing endothelial cells to adapt to acute changes in their
environment.
Mutator phenotypes of yeast strains heterozygous for mutations in the
MSH
2
gene
Authors: Drotschmann Karin , Clark Alan B. , Tran Hiep T. , Resnick Michael A. , Gordenin Dmitry A. , Kunkel Thomas A.
Published: July 2002

Heterozygosity for germ-line mutations in the DNA mismatch repair gene
MSH2
predisposes humans to cancer. Here we use a highly sensitive reporter to describe a spontaneous mutator phenotype in diploid yeast cells containing a deletion of only one
MSH2
allele. We also identify five
MSH2
missense mutations that have dominant mutator effects in heterozygous cells when expressed at normal levels from the natural
MSH2
promoter. For example, a 230-fold mutator effect is observed in an
MSH2
/
msh2
diploid strain in which Gly
693
, which is invariant in MutS homologs and involved in ATP hydrolysis, is changed to alanine. DNA binding data suggest that mismatch repair is suppressed by binding of a mutant Msh2–Msh6 heterodimer to a mismatch with subsequent inability to dissociate from the mismatch in the presence of ATP. A dominant mutator effect also is observed in yeast when Gly
693
is changed to serine. An early onset colorectal tumor is heterozygous for the analogous Gly → Ser mutation in
hMSH2
, and a second
hMSH2
mutation was not found, suggesting that this missense mutation may predispose to cancer via a dominant mutator effect. The mutator effects of the deletion mutant and the Gly → Ala missense mutant in yeast
MSH2
are enhanced by heterozygosity for a missense mutation in DNA polymerase δ that reduces its proofreading activity but is not a mutator in the heterozygous state. The synergistic effects of heterozygosity for mutations in two different genes that act in series to correct replication errors may be relevant to cancer predisposition.
Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization
Authors: Mateu Mauricio G. , Fersht Alan R.
Published: July 2002

We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.
Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex
Authors: Venkataraman Ganesh , Raman Rahul , Sasisekharan V. , Sasisekharan Ram
Published: July 2002

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.
The membrane-attached electron carrier cytochrome
c
y
from
Rhodobacter sphaeroides
is functional in respiratory but not in photosynthetic electron transfer
Authors: Myllykallio Hannu , Zannoni Davide , Daldal Fevzi
Published: July 2002

Rhodobacter
species are useful model organisms for studying the structure and function of
c
type cytochromes (Cyt
c
), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species,
Rhodobacter capsulatus
has a periplasmic Cyt
c
2

Rc

and a membrane-bound bipartite Cyt
c
y

Rc

. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently,
Rhodobacter sphaeroides
was thought to have only one of these two cytochromes, the soluble Cyt
c
2

Rs

. Recent work indicated that this species has a gene,

cycY
Rs

, that is highly homologous to

cycY
Rc

, and in the work presented here, functional properties of its gene product (Cyt
c
y

Rs

) are defined. It was found that Cyt
c
y

Rs

is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its
R. capsulatus
counterpart, Cyt
c
y

Rc

. Chimeric constructs have shown that the photosynthetic incapability of Cyt
c
y

Rs

is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt
c
oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?
Noninvasive measurement of gene expression in skeletal muscle
Authors: Walter Glenn , Barton Elisabeth R. , Sweeney H. Lee
Published: July 2002

We have developed a noninvasive detection method for expression of viral-mediated gene transfer. A recombinant adenovirus was constructed by using the gene for arginine kinase (AK), which is the invertebrate correlate to the vertebrate ATP-buffering enzyme, creatine kinase. Gene expression was noninvasively monitored using
31
P-magnetic resonance spectroscopy (
31
P-MRS). The product of the AK enzyme, phosphoarginine (PArg), served as an MRS-visible reporter of AK expression. The recombinant adenovirus coding for arginine kinase (rAdCMVAK) was injected into the right hindlimbs of neonatal mice. Two weeks after injection of rAdCMVAK, a unique
31
P-MRS resonance was observed. It was observable in all rAdCMVAK injected hindlimbs and was not present in the contralateral control or the vehicle injected limb. PArg and phosphocreatine (PCr) concentrations were calculated to be 11.6 ± 0.90 and 13.6 ± 1.1 mM respectively in rAdCMVAK injected limbs. AK activity was demonstrated
in vivo
by monitoring the decreases in PArg and ATP resonances during prolonged ischemia. After 1 h of ischemia intracellular pH was 6.73 ± 0.06, PCr/ATP was decreased by 77 ± 8%, whereas PArg/ATP was decreased by 50 ± 15% of basal levels. PArg and PCr returned to basal levels within 5 min of the restoration of blood flow. AK activity persisted for at least 8 mo after injection, indicating that adenoviral-mediated gene transfer can produce stable expression for long periods of time. Therefore, the cDNA encoding AK provides a useful reporter gene that allows noninvasive and repeated monitoring of gene expression after viral mediated gene transfer to muscle.
Reduced X-linked nucleotide polymorphism inDrosophila simulans
Authors: Begun David J. , Whitley Penn
Published: July 2002

Population genetic theory predicts that selectively driven changes of allele frequency for both beneficial and deleterious mutants reduce polymorphism at tightly linked sites. All else being equal, these reductions in polymorphism are expected to be greater when recombination rates are lower. Therefore, the empirical observation of a positive correlation between recombination rates and amounts of DNA polymorphism across theDrosophila melanogastergenome can be explained by two very different types of natural selection. Here, we evaluate alternative models of effects of selection on linked sites by comparison of X-linked and autosomal variation. We present polymorphism data from 40 genes distributed across chromosome arms X and 3R ofDrosophila simulans, a sibling species ofD. melanogaster. We find significantly less silent polymorphism inD. simulanson the X chromosome than on 3R, but no difference between arms for silent divergence between species. This pattern is incompatible with predictions from theoretical studies on the effect of negative selection on linked sites. We propose that some form of positive selection having greater effects on sex chromosomes than on autosomes is the better explanation for theD. simulansdata.
Genetic susceptibility to breast cancer: HLA DQB*03032 and HLA DRB1*11 may represent protective alleles
Authors: Chaudhuri Subhra , Cariappa Annaiah , Tang Mei , Bell Daphne , Haber Daniel A. , Isselbacher Kurt J. , Finkelstein Dianne , Forcione David , Pillai Shiv
Published: July 2002

Tumors are believed to emerge only when immune surveillance fails.
We wished to ascertain whether the failure to inherit putative
protective alleles of HLA class II genes is linked to the development
of breast cancer. We molecularly typed HLA DPB1, DQB1, DRB1, and DRB3
alleles in 176 Caucasian women diagnosed with early-onset breast cancer
and in 215 ethnically matched controls. HLA DQB*03032 was identified in
7% of controls but in no patients with early-onset breast cancer
(
P
= 0.0001). HLA DRB1*11 alleles were also
significantly overrepresented (
P
< 0.0001) in
controls (16.3%) as compared with patients with early-onset breast
cancer (3.5%). HLA DQB*03032 and HLA DRB1*11 alleles may have a
protective role in human breast cancer.
Changing T cell specificity by retroviral T cell receptor display
Authors: Kessels Helmut W. H. G. , van den Boom Marly D. , Spits Hergen , Hooijberg Erik , Schumacher Ton N. M.
Published: July 2002

The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generatedin vitroand introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequentin vitroselection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resultingin vitroselected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than thein vivogenerated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes.
Mice reconstituted with DNA polymerase β-deficient fetal liver cells are able to mount a T cell-dependent immune response and mutate their Ig genes normally
Authors: Esposito Gloria , Texido Gemma , Betz Ulrich A. K. , Gu Hua , Müller Werner , Klein Ulf , Rajewsky Klaus
Published: July 2002

The ubiquitously expressed, error-prone DNA polymerase β (polβ) plays a role in base excision repair, and the involvement of this molecule in the nonhomologous end joining (NHEJ) process of DNA repair has recently been demonstrated in yeast. Polβ-deficient mice are not viable, and studies on conditional mutants revealed a competitive disadvantage of polβ−/−vs. wild-type cells. We show here that polβ-deficient mice survive up to day 18.5 postcoitum, but die perinatally; a circumstance that allowed the investigation of a potential role of polβ in lymphocyte development by transfer of fetal liver cells (FLC) derived from polβ−/−embryos into lethally irradiated hosts. FLC transfers using mutant cells lead to an almost normal reconstitution of the lymphocyte compartment, indicating that polβ-deficiency does not prevent V(D)J recombination, which is known to employ factors of the NHEJ pathway. Mice reconstituted with polβ−/−FLC mount a normal T cell-dependent immune response against the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). Moreover, germinal center B cells from NP-immunized reconstituted mice show normal levels and patterns of somatic point mutations in their rearranged antibody genes, demonstrating that polβ is not critically involved in somatic hypermutation.
Learning What Cannot Be by Failing Expectations
Authors: Johansson Christer
Published: July 2002

This article presents an explanation for the lack of some neuter gender
forms in an otherwise productive paradigm for the formation of neuter gender
adjectives. The explanation draws on a proper understanding of
(exemplar-based) analogy under some conditions. One condition is that the
problematic form resembles a free morpheme by the loss of a
morpheme/syllable boundary in its derivation. The second condition is that
the problematic forms tend to share some property (for example, referring to
a nonverifiable property such as internal (mental) states). Problematic
forms might thus mutually support other such forms by analogy and
similarity, and thus create a paradigmatic gap. It is argued that exemplars
(with context), and analogy between similar exemplars, necessarily restrict
and licence the use of derived forms.
Biogenesis of the Saccharomyces cerevisiae Mating Pheromone a-Factor
Authors: Chen Peng , Sapperstein Stephanie K. , Choi Jonathan D. , Michaelis Susan
Published: July 2002

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification, NH2-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDSPAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.
Centromere Protein B Null Mice are Mitotically and Meiotically Normal but Have Lower Body and Testis Weights
Authors: Hudson Damien F. , Fowler Kerry J. , Earle Elizabeth , Saffery Richard , Kalitsis Paul , Trowell Helen , Hill Joanne , Wreford Nigel G. , de Kretser David M. , Cancilla Michael R. , Howman Emily , Hii Linda , Cutts Suzanne M. , Irvine Danielle V. , Choo K.H.A.
Published: July 2002

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.
δ-catenin, an Adhesive Junction–associated Protein Which Promotes Cell Scattering
Authors: Lu Qun , Paredes Mercedes , Medina Miguel , Zhou Jianhua , Cavallo Robert , Peifer Mark , Orecchio Lisa , Kosik Kenneth S.
Published: July 2002

The classical adherens junction that holds epithelial cells together consists of a protein complex in which members of the cadherin family linked to various catenins are the principal components. δ-catenin is a mammalian brain protein in the Armadillo repeat superfamily with sequence similarity to the adherens junction protein p120ctn. We found that δ-catenin can be immunoprecipitated as a complex with other components of the adherens junction, including cadherin and β-catenin, from transfected cells and brain. The interaction with cadherin involves direct contact within the highly conserved juxtamembrane region of the COOH terminus, where p120ctn also binds. In developing mouse brain, staining with δ-catenin antibodies is prominent towards the apical boundary of the neuroepithelial cells in the ventricular zone. When transfected into Madin-Darby canine kidney (MDCK) epithelial cells δ-catenin colocalized with cadherin, p120ctn, and β-catenin. The Arm domain alone was sufficient for achieving localization and coimmunoprecipitation with cadherin. The ectopic expression of δ-catenin in MDCK cells altered their morphology, induced the elaboration of lamellipodia, interfered with monolayer formation, and increased scattering in response to hepatocyte growth factor treatment. We propose that δ-catenin can regulate adhesion molecules to implement the organization of large cellular arrays necessary for tissue morphogenesis.