bluuebunny/crossref_metadata_2025 huggingface dataset:
Triplet repeat polymorphism in the transmembrane region of the MICA gene: A strong association of six GCT repetitions with Behçet disease
Authors: Mizuki Nobuhisa , Ota Masao , Kimura Minoru , Ohno Shigeaki , Ando Hitoshi , Katsuyama Yoshihiko , Yamazaki Masaaki , Watanabe Koji , Goto Kaori , Nakamura Satoshi , Bahram Seiamak , Inoko Hidetoshi
Published: July 2002

A member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), MICA, has been recently identified near the HLA-B gene on the short arm of human chromosome 6. The predicted amino acid sequence of the MICA chain suggests that it folds similarly to typical class I chains and may have the capacity to bind peptides or other short ligands. Therefore, MICA is predicted to have a specialized function in antigen presentation or T cell recognition. During nucleotide sequence analyses of the MICA genomic clone, we found a triplet repeat microsatellite polymorphism of (GCT/AGC)
n
in the transmembrane (TM) region of the MICA gene. In 68 HLA homozygous B cell lines, 5 distinct alleles of this microsatellite sequence were detected. One of them contained an additional one base insertion that created a frameshift mutation resulting in a premature termination codon in the TM region. This particular allele may encode a soluble, secreted form of the MICA molecule. In addition, we have investigated this microsatellite polymorphism in 77 Japanese patients with Behçet disease, which is known to be associated with HLA-B51. The microsatellite allele consisting of 6 repetitions of GCT/AGC was present at significantly higher frequency in the patient group (
Pc
= 0.00055) than in a control population. Furthermore, the (GCT/AGC)
6
allele was present in all B51 positive patients and in an additional 13 B51 negative patients. These results suggest the possibility of a primary association of Behçet disease with MICA rather than HLA-B.
Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors
Authors: Koeberl Dwight D. , Alexander Ian E. , Halbert Christine L. , Russell David W. , Miller A. Dusty
Published: July 2002

We previously found that gene transduction by adeno-associated virus (AAV) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors. Here we show that previous γ-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. Similar rates of hepatic transduction were obtained by direct injection of the liver or by systemic delivery via tail vein injection. Hepatocytes were much more efficiently transduced than other cells after systemic delivery, and up to 3% of all hepatocytes could be transduced after one vector injection. The presence of wild-type AAV, which contaminates many AAV vector preparations, was required to observe a full response to γ-irradiation. Injection of mice with AAV vectors encoding human clotting factor IX after γ-irradiation resulted in synthesis of low levels of human clotting factor IX for the 5-month period of observation. These studies show the potential of targeted gene transduction of the liver by AAV vectors for treatment of various hematological or metabolic diseases.
Positive selection of HIV-1 cytotoxic T lymphocyte escape variants during primary infection
Authors: Price David A. , Goulder Philip J. R. , Klenerman Paul , Sewell Andrew K. , Easterbrook Philippa J. , Troop Maxine , Bangham Charles R. M. , Phillips Rodney E.
Published: July 2002

Cytotoxic T lymphocytes (CTLs) are thought to play a crucial role in the termination of the acute primary HIV-1 syndrome, but clear evidence for this presumption has been lacking. Here we demonstrate positive selection of HIV-1 proviral sequences encoding variants within a CTL epitope in Nef, a gene product critical for viral pathogenicity, during and after seroconversion. These positively selected HIV-1 variants carried epitope sequence changes that either diminished or escaped CTL recognition. Other proviruses had mutations that abolished the Nef epitope altogether. These results provide clear evidence that CTLs exert selection pressure on the viral population in acute HIV-1 infection.
Functional characterization of
ETV6
and
ETV6/CBFA
2 in the regulation of the
MCSFR
proximal promoter
Authors: Fears S. , Gavin M. , Zhang D.-E. , Hetherington C. , Ben-David Y. , Rowley J. D. , Nucifora G.
Published: July 2002

The
ETV6/CBFA2
(
TEL/AML1
) fusion gene occurs as a result of the chromosome translocation t(12;21)(p13;q22) in up to 30% of children diagnosed with B cell precursor (cd10
+
, cd19
+
) acute lymphoblastic leukemia. Leukemic cells that have acquired the t(12;21) usually demonstrate loss of the remaining normal
ETV6
(
TEL
) allele. Using reporter gene assays we have functionally characterized both the normal ETV6 and ETV6/CBFA2 fusion proteins in the regulation of the
MCSFR
proximal promoter. Neither ETV6 or ETV6/CBFA2 has any significant, detectable effect on the promoter by itself. However, both ETV6 and ETV6/CBFA2 inhibit the activation of the promoter by CBFA2B(AML1B) and C/EBPa. We have shown that a 29-bp region of the
MCSFR
promoter containing the binding sites for CBFA2B and C/EBPa is sufficient for the inhibition by ETV6 and ETV6/CBFA2. Mutational analysis of the
MCSFR
promoter revealed that binding of both CBFA2B and C/EBPa to their respective sites is necessary for the inhibition by ETV6 and ETV6/CBFA2. Deletion of the helix–loop–helix (HLH) region from the cDNAs of
ETV6
and
ETV6/CBFA2
decreased but did not completely abrogate the ability of either construct to inhibit promoter activation. We also found that the ETS DNA binding region of ETV6 is necessary for inhibition of the promoter. Addition of ETS1 and FLI1, two ETS family members that have homology in the 5′ HLH region, but not Spi1, an ETS family member without the 5′ HLH region, also inhibited reporter gene expression. Our data show that the inhibition mediated by ETV6 and ETV6/CBFA2, in the context of the
MCSFR
promoter, depend on interactions with other proteins, not just CBFA2B. Our results also indicate that the transactivation characteristics of ETV6/CBFA2 are a combination of positive and negative regulatory properties.
Peptide inhibition of glomerular deposition of an anti-DNA antibody
Authors: Gaynor Bruce , Putterman Chaim , Valadon Philippe , Spatz Linda , Scharff Matthew D. , Diamond Betty
Published: July 2002

Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit in the kidneys of lupus patients to cause glomerulonephritis. Recent data suggest that a significant proportion of anti-DNA antibodies may cross-react with renal antigens and be sequestered in the kidney by virtue of this cross-reactivity. If this is true, antigenic competition for pathogenic antibodies might prevent their deposition in kidneys and the ensuing tissue damage. To generate surrogate antigens that could be used for this purpose, we have used peptide display phage libraries to identify peptides that react with R4A, a pathogenic mouse monoclonal anti-DNA antibody that deposits in glomeruli. We have demonstrated that the peptides bind in or near the double-stranded DNA binding site. Furthermore, the peptides are bound preferentially by the R4A antibody as compared with two closely related antibodies derived from it, one of which deposits in renal tubules and one of which displays no renal pathogenicity. Administration of one of these peptides in a soluble form protects mice from renal deposition of the R4A anti-DNA antibodyin vivo. This represents a new therapeutic approach in systemic lupus erythematosus that focuses on protecting target organs from antibody mediated injury.
EEN
encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to
MLL
in human leukemia
Authors: So Chi Wai , Caldas Carlos , Liu Meng-Min , Chen Sai-Juan , Huang Qiu-Hua , Gu Long-Jun , Sham Mai Har , Wiedemann Leanne Marie , Chan Li Chong
Published: July 2002

The
MLL
gene, the closest human homologue to the
Drosophila
trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new partner gene,
EEN
, fused to
MLL
in a case of acute myeloid leukemia. The gene is located on chromosome 19p13, where two other
MLL
partner genes,
ENL
and
ELL
/
MEN
have also been identified. The deduced protein of 368 aa contains a central α-helical region and a C-terminal Src homology 3 (SH3) domain most similar to the C-terminal SH3 domain found in the
Grb2/Sem-5/Drk
family of genes. Sequence analysis of the fusion
MLL
/
EEN
transcript in our patient reveals that exon 6 of
MLL
is fused to the N-terminal end of
EEN
, a fusion that would create a chimeric protein that includes the major functional domain of
EEN
.
EEN
is expressed in a variety of tissue types and encodes a protein of approximately 46 kDa. The
EEN
protein is the human homologue of a member of a recently described murine SH3 domain-containing protein family. It is also highly related to a putative gene identified in
Caenorhabditis elegans
, and a number of similar sequences are present in the EST databases of several species.
Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice
Authors: Schlaeger Thorsten M. , Bartunkova Sona , Lawitts Joel A. , Teichmann Gunnar , Risau Werner , Deutsch Urban , Sato Thomas N.
Published: July 2002

TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5′ flanking region of the
TIE2
promoter is capable of directing β-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse
TIE2
gene. Furthermore, combination of the
TIE2
promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an
in vivo
expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs
in vivo
. Furthermore, we were able to identify a short region critical for enhancer function
in vivo
that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the
TIE2
gene.
Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts
Authors: Kumar Madhur , Carmichael Gordon G.
Published: July 2002

Antisense RNA may regulate the expression of a number of eukaryotic genes, but little is known about its prevalence or mechanism of action. We have used a model system in which antisense control can be studied both genetically and biochemically. Late in polyoma virus infection, early-strand mRNA levels are down-regulated by nuclear antisense RNA from the late strand. Analysis of early-strand transcripts isolated late in infection revealed extensive base modifications. In many transcripts almost half of the adenosines were altered to inosines or guanosines. These results suggest modification of RNA duplexes by double-stranded RNA adenosine deaminase or a related enzyme. Probes that detect only modified RNAs revealed that these molecules are not highly unstable, but accumulate within the nucleus and are thus inert for gene expression. Antisense-induced modifications can account for most or all of the observed regulation, with the lowered levels of early-strand RNAs commonly observed late in infection resulting from the fact that many transcripts are invisible to standard hybridization probes. This work suggests that similar antisense-mediated control mechanisms may also operate under physiological conditions in uninfected eukaryotic cells, and leads to the proposal that there is a novel pool of nuclear RNAs that cannot be seen with many molecular probes heretofore used.
The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus
Authors: Nickerson Jeffrey A. , Krockmalnic Gabriela , Wan Katherine M. , Penman Sheldon
Published: July 2002

The nucleus is an intricately structured integration of many functional domains whose complex spatial organization is maintained by a nonchromatin scaffolding, the nuclear matrix. We report here a method for preparing the nuclear matrix with improved preservation of ultrastructure. After the removal of soluble proteins, the structures of the nucleus were extensively cross-linked with formaldehyde. Surprisingly, the chromatin could be efficiently removed by DNase I digestion leaving a well preserved nuclear matrix. The nuclear matrix uncovered by this procedure consisted of highly structured fibers, connected to the nuclear lamina and built on an underlying network of branched 10-nm core filaments. The relative ease with which chromatin and the nuclear matrix could be separated despite extensive prior cross-linking suggests that there are few attachment points between the two structures other than the connections at the bases of chromatin loops. This is an important clue for understanding chromatin organization in the nucleus.
The age of the universe from nuclear chronometers
Authors: Truran J. W.
Published: July 2002

An overview is presented of the current situation regarding
radioactive dating of the matter of which our Galaxy is comprised. A
firm lower bound on the age from nuclear chronometers of ≈9–10 Gyr
is entirely consistent with age determinations from globular clusters
and white dwarf cooling histories. The reasonable assumption of an
approximately uniform nucleosynthesis rate yields an age for the Galaxy
of 12.8 ± 3 Gyr, which again is consistent with current
determinations from other methods.
A mitochondrial-like chaperonin 60 gene in
Giardia lamblia:
Evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria
Authors: Roger Andrew J. , Svärd Staffan G. , Tovar Jorge , Clark C. Graham , Smith Michael W. , Gillin Frances D. , Sogin Mitchell L.
Published: July 2002

Diplomonads, parabasalids, as represented by trichomonads, and microsporidia are three protist lineages lacking mitochondria that branch earlier than all other eukaryotes in small subunit rRNA and elongation factor phylogenies. The absence of mitochondria and plastids in these organisms suggested that they diverged before the origin of these organelles. However, recent discoveries of mitochondrial-like heat shock protein 70 and/or chaperonin 60 (cpn60) genes in trichomonads and microsporidia imply that the ancestors of these two groups once harbored mitochondria or their endosymbiotic progenitors. In this report, we describe a mitochondrial-like cpn60 homolog from the diplomonad parasite
Giardia lamblia.
Northern and Western blots reveal that the expression of cpn60 is independent of cellular stress and, except during excystation, occurs throughout the
G. lamblia
life cycle. Phylogenetic analyses position the
G. lamblia
cpn60 in a clade that includes mitochondrial and hydrogenosomal cpn60 proteins. The most parsimonious interpretation of these data is that the cpn60 gene was transferred from the endosymbiotic ancestors of mitochondria to the nucleus early in eukaryotic evolution, before the divergence of the diplomonads and trichomonads from other extant eukaryotic lineages. A more complicated explanation requires that these genes originated from distinct α-proteobacterial endosymbioses that formed transiently within these protist lineages.
Obesity and hyperleptinemia in metallothionein (-I and -II) null mice
Authors: Beattie J. H. , Wood A. M. , Newman A. M. , Bremner I. , Choo K. H. A. , Michalska A. E. , Duncan J. S. , Trayhurn P.
Published: July 2002

Metallothionein (MT) has several putative roles in metal detoxification, in Zn and Cu homeostasis, in scavenging free radicals, and in the acute phase response. Mice of mixed 129/Ola and C57BL/6J background with targeted disruption of MT-I and MT-II genes are more sensitive to toxic metals and oxidative stress. We noted that these animals were larger than most strains of mice, and we systematically studied aspects of their physiology and biochemistry relating to energy metabolism. During the first 2 weeks after weaning, the growth rates of MT-null and C57BL/6J mice were similar, but the transgenic mice became significantly heavier at age 5–6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with C57BL/6J mice. Most 22- to 39-week-old male MT-null mice were obese, as shown by increased fat accretion, elevated obese (
ob
) gene expression, and high plasma leptin levels, similar to those recorded in Zucker fatty (
fa/fa
) rats. Seven-week-old MT-null mice also had significantly higher levels of plasma leptin and elevated expression of
ob
, lipoprotein lipase, and CCAAT enhancer binding protein α genes as compared with age-matched C57BL/6J mice. These observations indicate that abnormal accretion of body fat and adipocyte maturation is initiated at 5–7 weeks of age, possibly coincident with sexual maturation. Targeted disruption of MT-I and MT-II genes seems to induce moderate obesity, providing a new obese animal model. A link between MT and the regulation of energy balance is implied.
Limit, logic, and computation
Authors: Freedman Michael H.
Published: July 2002

We introduce “ultrafilter limits” into the classical Turing model of computation and develop a paradigm for interpreting the problem of distinguishing the class
P
from
NP
as a logical problem of decidability. We use
P
(
NP
) to denote decision problems which can be solved on a (nondeterministic) Turing machine in polynomial time. The concept is that in an appropriate limit it may be possible to prove that problems in
P
are still decidable, so a problem whose limit is undecidable would be established as lying outside of
P
.
Three independent lines of evidence suggest retinoids as causal to schizophrenia
Authors: Goodman Ann B.
Published: July 2002

Retinoid dysregulation may be an important factor in the etiology
of schizophrenia. This hypothesis is supported by three independent
lines of evidence that triangulate on retinoid involvement in
schizophrenia: (
i
) congenital anomalies similar to those
caused by retinoid dysfunction are found in schizophrenics and their
relatives; (
ii
) those loci that have been suggestively
linked to schizophrenia are also the loci of the genes of the retinoid
cascade (convergent loci); and (
iii
) the transcriptional
activation of the dopamine D2 receptor and numerous schizophrenia
candidate genes is regulated by retinoic acid. These findings suggest a
close causal relationship between retinoids and the underlying
pathophysiological defects in schizophrenia. This leads to specific
strategies for linkage analyses in schizophrenia. In view of the
heterodimeric nature of the retinoid nuclear receptor transcription
factors, e.g., retinoid X receptor β at chromosome 6p21.3 and
retinoic acid receptor β at 3p24.3, two-locus linkage models
incorporating genes of the retinoid cascade and their heterodimeric
partners, e.g., peroxisome proliferator-activated receptor α at
chromosome 22q12-q13 or nuclear-related receptor 1 at chromosome
2q22-q23, are proposed. New treatment modalities using retinoid analogs
to alter the downstream expression of the dopamine receptors and other
genes that are targets of retinoid regulation, and that are thought to
be involved in schizophrenia, are suggested.
Broken symmetry and strangeness of the semiconductor impurity band metal-insulator transition
Authors: Phillips J. C.
Published: July 2002

The filamentary model of the metal-insulator transition in randomly doped semiconductor impurity bands is geometrically equivalent to similar models for continuous transitions in dilute antiferromagnets and even to the λ transition in liquid He, but the critical behaviors are different. The origin of these differences lies in two factors: quantum statistics and the presence of long range Coulomb forces on both sides of the transition in the electrical case. In the latter case, in addition to the main transition, there are two satellite transitions associated with disappearance of the filamentary structure in both insulating and metallic phases. These two satellite transitions were first identified by Fritzsche in 1958, and their physical origin is explained here in geometrical and topological terms that facilitate calculation of critical exponents.
Functional and molecular characterization of a glycosomal PP
i
-dependent enzyme in trypanosomatids: Pyruvate, phosphate dikinase
Authors: Bringaud Frederic , Baltz Dominique , Baltz Theo
Published: July 2002

Trypanosomatids are parasitic protists that have an ATP-dependent glycolysis with no indication of PP
i
-dependent metabolism. Most of the glycolysis takes place in peroxisome-like organelles, the glycosomes. We characterized in
Trypanosoma brucei
a single-copy gene encoding a PP
i
-dependent enzyme, pyruvate, phosphate dikinase (PPDK), which was expressed functionally in
Escherichia coli
. Specific antibodies detected a 100-kDa protein in procyclic forms but not in mammalian forms of
T. brucei
, indicating a differential expression. Glycosomal localization of PPDK was determined by immunofluorescence analysis and was confirmed by Western blot analysis on glycosomal fractions by using anti-PPDK antibodies. Expression and localization of recombinant PPDKs in procyclic forms of
T. brucei
showed that the AKL motif at the C-terminal extremity of PPDK is necessary for glycosomal targeting. PPDK was detected in every trypanosomatid tested—
Trypanosoma congolense
,
Trypanosoma vivax
,
Trypanosoma cruzi
,
Phytomonas
,
Crithidia
and
Leishmania
—with a good correlation between amount of protein and enzymatic activity. The precise role of PPDK in trypanosomatid carbohydrate metabolism remains to be clarified.
Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes
Authors: Zhang Lei , Wang Danher , Fischer Horst , Fan Pei-dong , Widdicombe Jonathan H. , Kan Yuet Wai , Dong Jian-yun
Published: July 2002

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.
Evidence for regulation of cartilage differentiation by the homeobox gene
Hoxc-8
Authors: Yueh Y. Gloria , Gardner David P. , Kappen Claudia
Published: July 2002

Homeobox genes of the
Hox
class are required for proper patterning of skeletal elements, but how they regulate the differentiation of specific tissues is unclear. We show here that overexpression of a
Hoxc-8
transgene causes cartilage defects whose severity depends on transgene dosage. The abnormal cartilage is characterized by an accumulation of proliferating chondrocytes and reduced maturation. Since
Hoxc-8
is normally expressed in chondrocytes, these results suggest that
Hoxc-8
continues to regulate skeletal development well beyond pattern formation in a tissue-specific manner, presumably by controlling the progression of cells along the chondrocyte differentiation pathway. The comparison to
Hoxd-4
and
Isl-1
indicates that this role in chondrogenesis is specific to proteins of the Hox class. Their capacity for regulation of cartilage differentiation suggests that
Hox
genes could also be involved in human chondrodysplasias or other cartilage disorders.
Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS
Authors: Gange Stephen J. , Muñoz Alvaro , Chmiel Joan S. , Donnenberg Albert D. , Kirstein Lynn M. , Detels Roger , Margolick Joseph B.
Published: July 2002

Studies of circulating T (CD3
+
) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5–2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log
10
CD3
+
cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3
+
lymphocyte counts before the inflection point was close to zero for HIV− and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3
+
cell counts relative to HIV− men and HIV+ men not developing AIDS. A CD3
+
cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.
Allelic association under map error and recombinational heterogeneity: A tale of two sites
Authors: Lonjou Christine , Collins Andrew , Ajioka Richard S. , Jorde Lynn B. , Kushner James P. , Morton Newton E.
Published: July 2002

Recombination acts on the genetic map, not on the physical map. On the other hand, the physical map is usually more accurate. Choice of the genetic or physical map for positional cloning by allelic association depends on the goodness of fit of data to each map under an established model. Huntington disease illustrates the usual case in which the greater reliability of physical data outweighs recombinational heterogeneity. Hemochromatosis represents an exceptional case in which unrecognized recombinational heterogeneity retarded positional cloning for a decade. The Malecot model performs well for major genes, but no approach assuming either equilibrium or disequilibrium has been validated for oligogenes contributing to common disease. In this case of greatest interest, the power of allelic association relative to linkage is less clear than for major genes.
Tests and estimates of allelic association in complex inheritance
Authors: Morton N. E. , Collins A.
Published: July 2002

Family-based procedures such as the transmission disequilibrium test (TDT) were motivated by concern that sample-based methods to map disease genes by allelic association are not robust to population stratification, migration, and admixture. Other factors to consider in designing a study of allelic association are specification of gene action in a weakly parametric model, efficiency, diagnostic reliability for hypernormal individuals, interest in linkage and imprinting, and sibship composition. Family-based samples lend themselves to the TDT despite its inefficiency compared with cases and unrelated normal controls. The TDT has an efficiency of 1/2 for parent-offspring pairs and 2/3 for father-mother-child trios. Against cases and hypernormal controls, the efficiency is only 1/6 on the null hypothesis. Although dependent on marker gene frequency and other factors, efficiency for hypernormal controls is always greater than for random controls. Efficiency of the TDT is increased in multiplex families and by inclusion of normal sibs, approaching a case-control design with normal but not hypernormal controls. Isolated cases favor unrelated controls, and only in exceptional populations would avoidance of stratification justify a family-based design to map disease genes by allelic association.
BCL-6 mutations in normal germinal center B cells: Evidence of somatic hypermutation acting outside Ig loci
Authors: Pasqualucci Laura , Migliazza Anna , Fracchiolla Nicola , William Christopher , Neri Antonino , Baldini Luca , Chaganti R. S. K. , Klein Ulf , Küppers Ralf , Rajewsky Klaus , Dalla-Favera Riccardo
Published: July 2002

The molecular mechanism involved in the process of antigen-driven somatic hypermutation of Ig genes is unknown, but it is commonly believed that this mechanism is restricted to the Ig loci. B cell lymphomas commonly display multiple somatic mutations clustering in the 5′-regulatory region of BCL-6, a proto-oncogene encoding for a POZ/Zinc finger transcriptional repressor expressed in germinal center (GC) B cells and required for GC formation. To determine whether BCL-6 mutations represent a tumor-associated phenomenon or reflect a physiologic mechanism, we screened single human tonsillar GC B cells for mutations occurring in the BCL-6 5′-noncoding region and in the Ig variable heavy chain sequences. Thirty percent of GC B cells, but not naive B cells, displayed mutations in the 742 bp region analyzed within the first intron of BCL-6 (overall frequency: 5 × 10−4/bp). Accordingly, an expanded survey in lymphoid malignancies showed that BCL-6 mutations are restricted to B cell tumors displaying GC or post-GC phenotype and carrying mutated Ig variable heavy chain sequences. These results indicate that the somatic hypermutation mechanism active in GC B cells physiologically targets non-Ig sequences.
Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses
Authors: Xu Jin-Rong , Staiger Christopher J. , Hamer John E.
Published: July 2002

The rice blast fungus,
Magnaporthe grisea,
generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of
slt2
null mutants. The
mps1–1
Δ mutants of
M. grisea
have some phenotypes in common with
slt2
mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly,
mps1–1
Δ mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although
mps1–1
Δ mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that
MPS1
is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.
Nucleotide-dependent conformational change near the fulcrum region in
Dictyostelium
myosin II
Authors: Liang Wenchuan , Spudich James A.
Published: July 2002

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin–myosin–ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from
Dictyostelium
in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1–SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in
Dictyostelium
myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1–SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the
Dictyostelium
myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.
Chemical synthesis of the precursor molecule of the
Aequorea
green fluorescent protein, subsequent folding, and development of fluorescence
Authors: Nishiuchi Yuji , Inui Tatsuya , Nishio Hideki , Bódi József , Kimura Terutoshi , Tsuji Frederick I. , Sakakibara Shumpei
Published: July 2002

The present paper describes the total chemical synthesis of the precursor molecule of the
Aequorea
green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λ
max
= 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.
A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and inEscherichia coli
Authors: Spinelli Sherry L. , Malik Harmit S. , Consaul Sandra A. , Phizicky Eric M.
Published: July 2002

tRNA splicing in the yeastSaccharomyces cerevisiaerequires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2′-phosphotransferase to transfer the splice junction 2′-phosphate from ligated tRNA to NAD, producing ADP ribose 1′′–2′′ cyclic phosphate (Appr>p). We show here that functional 2′-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicansandSchizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2′-phosphotransferase is found also in the bacteriumEscherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2′-phosphate. Analysis of the database shows that likely members of the 2′-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidusandPyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2′-phosphotransferase into Eubacteria, suggesting that the 2′-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2′-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential inE. coli, the continued presence of 2′-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.
Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels
Authors: Shapiro Mark S. , Zagotta William N.
Published: July 2002

Native cylic nucleotide-gated (CNG) channels are composed of α and β subunits. Olfactory CNG channels were expressed from rat cDNA clones in
Xenopus
oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the α and β subunits. Dimers contained three subunit types: α
wt
, β
wt
, and α
m
. The α
m
subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of α
wt
α
wt
dimers and from α
wt
monomers were indistinguishable. Channels from injection of α
wt
α
m
dimers had apparent affinities 3-fold lower than α
wt
homomultimers, suggesting a channel with two α
wt
and two α
m
subunits. Channels from coinjection of α
wt
α
wt
and ββ dimers were indistinguishable from those composed of α and β monomers and shared all of the characteristics of the α+β phenotype of heteromeric channels. Coinjection of α
wt
α
m
and ββ dimers yielded channels also of the α+β phenotype but with an apparent affinity 3-fold lower, indicating the presence of α
m
in the tetramer and that α+β channels have adjacent α-subunits. To distinguish between an α-α-α-β and an α-α-β-β arrangement, we compared apparent affinities for channels from coinjection of α
wt
α
wt
and βα
wt
or α
wt
α
wt
and βα
m
dimers. These channels were indistinguishable. To further argue against an α-α-α-β arrangement, we quantitatively compared dose–response data for channels from coinjection of α
wt
α
m
and ββ dimers to those from α and β monomers. Taken together, our results are most consistent with an α-α-β-β arrangement for the heteromeric olfactory CNG channel.
WISP
genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors
Authors: Pennica Diane , Swanson Todd A. , Welsh James W. , Roy Margaret A. , Lawrence David A. , Lee James , Brush Jennifer , Taneyhill Lisa A. , Deuel Bethanne , Lew Michael , Watanabe Colin , Cohen Robert L. , Melhem Mona F. , Finley Gene G. , Quirke Phil , Goddard Audrey D. , Hillan Kenneth J. , Gurney Austin L. , Botstein David , Levine Arnold J.
Published: July 2002

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes,
WISP-1
and
WISP-2
, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene,
WISP-3
, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated
WISP
induction to be associated with the expression of Wnt-1. These included (
i
) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (
ii
) Wnt-1 transgenic mice. The
WISP-1
gene was localized to human chromosome 8q24.1–8q24.3.
WISP-1
genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa.
WISP-3
mapped to chromosome 6q22–6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast,
WISP-2
mapped to human chromosome 20q12–20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the
WISP
genes may be downstream of Wnt-1 signaling and that aberrant levels of
WISP
expression in colon cancer may play a role in colon tumorigenesis.
Amber suppression in
Escherichia coli
by unusual mitochondria-like transfer RNAs
Authors: Bourdeau Véronique , Steinberg Sergey V. , Ferbeyre Gerardo , Emond Rémi , Cermakian Nicolas , Cedergren Robert
Published: July 2002

The “cloverleaf” base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be
bona fide
alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons
in vivo
.
Protein kinase A directly regulates the activity and proteolysis of
cubitus interruptus
Authors: Chen Yang , Gallaher Nicole , Goodman Richard H. , Smolik Sarah M.
Published: July 2002

Cubitus interruptus
(
Ci
) is a transcriptional factor that is positively regulated by the
hedgehog
(
hh
) signaling pathway. Recent work has shown that a 75-kDa proteolytic product of the full-length CI protein translocates to the nucleus and represses the transcription of CI target genes. In cells that receive the
hh
signal, the proteolysis of CI is inhibited and the full-length protein can activate the
hh
target genes. Because protein kinase A (PKA) inhibits the expression of the
hh
target genes in developing embryos and discs and the loss of PKA activity results in elevated levels of full-length CI protein, PKA might be involved directly in the regulation of CI proteolysis. Here we demonstrate that the PKA pathway antagonizes the
hh
pathway by phosphorylating CI. We show that the PKA-mediated phosphorylation of CI promotes its proteolysis from the full-length active form to the 75-kDa repressor form. The PKA catalytic subunit increases the proteolytic processing of CI and the PKA inhibitor, PKI, blocks the processing. In addition, cells do not process the CI protein to the 75-kDa repressor when all of the PKA sites in CI are mutated. Mutant CI proteins that cannot be phosphorylated by PKA have increased transcriptional activity compared with wild-type CI. In addition, exogenous PKA can increase further the transcriptional activity of the CI mutant, suggesting that PKA has a second positive, indirect effect on CI activity. In summary, we show that the modulation of the
hh
signaling pathway by PKA occurs directly at the level of CI phosphorylation.
Subcellular localization of mineralocorticoid receptors in living cells: Effects of receptor agonists and antagonists
Authors: Fejes-Tóth Géza , Pearce David , Náray-Fejes-Tóth Anikó
Published: July 2002

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green fluorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclear-to-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were retained in nuclear matrix preparations after
in vivo
crosslinking. These data strongly suggest that hormone-activated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids.