bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.
Authors: Sage E , Lamolet B , Brulay E , Moustacchi E , Chteauneuf A , Drobetsky E A
Published: July 2002

To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cells. The frequency distributions of mutational classes induced by UVB and by simulated sunlight in repair-deficient CHO cells were virtually identical, each showing a marked increase in tandem CC-->TT transitions relative to NER-proficient cells. A striking increase in CC-->TT events was also previously documented for mutated p53 tumor-suppressor genes from nonmelanoma tumors of NER-deficient, skin cancer-prone xeroderma pigmentosum patients, compared to normal individuals. The data therefore indicate that the aprt gene in NER-deficient cultured rodent cells irradiated with artificial solar light generates the same distinctive "fingerprint" for sunlight mutagenesis as the p53 locus in NER-deficient humans exposed to natural sunlight in vivo. Moreover, in strong contrast to the situation for repair-component CHO cells, where a significant role for UVA was previously noted, the mutagenic specificity of simulated sunlight in NER-deficient CHO cells and of natural sunlight in humans afflicted with xeroderma pigmentosum can be entirely accounted for by the UVB portion of the solar wavelength spectrum.
Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.
Authors: Clark T G , Lin T L , Dickerson H W
Published: July 2002

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.
P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.
Authors: Szeto D P , Ryan A K , O'Connell S M , Rosenfeld M G
Published: July 2002

A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in the mature gland. P-OTX is able to independently activate and to synergize with Pit-1 on pituitary-specific target gene promoters. Therefore, P-OTX may subserve functions in generating both precursor and specific cell phenotypes in the anterior pituitary gland and in several other organs.
Pinus banksiana
has at least seven expressed alcohol dehydrogenase genes in two linked groups
Authors: Perry Daniel J. , Furnier Glenn R.
Published: July 2002

The alcohol dehydrogenase (
Adh
) gene family is much
more complex in
Pinus banksiana
than in angiosperms,
with at least seven expressed genes organized as two tightly linked
clusters. Intron number and position are highly conserved between
P. banksiana
and angiosperms. Unlike angiosperm
Adh
genes, numerous duplications, as large as 217 bp,
were observed within the noncoding regions of
P. banksiana
Adh
genes and may be a common feature of conifer genes. A high
frequency of duplication over a wide range of scales may contribute to
the large genome size of conifers.
PCR-based development of DNA substrates containing modified bases: An efficient system for investigating the role of the exocyclic groups in chemical and structural recognition by minor groove binding drugs and proteins
Authors: Bailly Christian , Payet Dominique , Travers Andrew A. , Waring Michael J.
Published: July 2002

DNA molecules containing inosine in place of guanosine and/or
2,6-diaminopurine in place of adenine have been synthesized and tested
as substrates for binding of sequence-selective ligands, both small and
large. Footprinting patterns reveal that the binding sites for AT- or
GC-specific antibiotics (distamycin or mithramycin, respectively) are
completely changed in the modified DNAs, as expected for direct
sequence readout involving contact with the purine 2-amino group.
However, we also find large changes in the binding of HMG-D, a member
of the HMG-1 family of chromosomal proteins, pointing to an indirect
influence of the exocyclic amino group on ligand binding via an effect
on the deformability of the double helix. This interpretation is
confirmed by the finding that deoxyuridine-containing poly- and
oligonucleotides, which lack the exocyclic methyl group of thymidine in
the major groove, interact 5–10 times more strongly with HMG-D than do
their counterparts containing natural nucleotides.
The
Saccharomyces CDC
13
protein is a single-strand TG
1–3
telomeric DNA-binding protein
in vitro
that affects telomere behavior
in vivo
Authors: Lin Jing-Jer , Zakian Virginia A.
Published: July 2002

Saccharomyces
telomeres consist of ≈300 bp of
C
1–3
A/TG
1–3
DNA. Cells lacking the activity
of the essential gene
CDC13
display a cell cycle arrest
mediated by the DNA damage sensing,
RAD9
cell cycle
checkpoint, presumably because they exhibit strand-specific loss of
telomeric and telomere-adjacent DNA [Garvik, B., Carson, M. &
Hartwell, L. (1995)
Mol. Cell. Biol.
15, 6128–6138].
Cdc13p expressed in
Escherichia coli
or overexpressed in
yeast bound specifically to single-strand TG
1–3
DNA. The
specificity of binding displayed by Cdc13p
in vitro
indicates that
in vivo
it could bind to both the short,
constitutive single-strand TG
1–3
tails thought to be
present at telomeres at most times in the cell cycle as well as to the
long single-strand TG
1–3
tails that are intermediates in
telomere replication. Genes located near yeast telomeres are
transcriptionally repressed, a phenomenon known as telomere position
effect. Cells overexpressing a mutant form of Cdc13p had reduced
telomere position effect at high temperatures. These data suggest that
Cdc13p functions by binding directly to telomeric DNA, thereby limiting
its accessibility to degradation and transcription as well as masking
it from factors that detect damaged DNA.
Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus
Authors: Grossman Zvi , Singer Alfred
Published: July 2002

Immature CD4+CD8+thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulationper se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other.
Ca
2+
/calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation
Authors: Nayak A. S. , Moore C. I. , Browning M. D.
Published: July 2002

Long-term potentiation (LTP) is an increase in synaptic
responsiveness thought to be involved in mammalian learning and memory.
The localization (presynaptic and/or postsynaptic) of changes
underlying LTP has been difficult to resolve with current
electrophysiological techniques. Using a biochemical approach, we have
addressed this issue and attempted to identify specific molecular
mechanisms that may underlie LTP. We utilized a novel
multiple-electrode stimulator to produce LTP in a substantial portion
of the synapses in a hippocampal CA1 minislice and tested the effects
of such stimulation on the presynaptic protein synapsin I. LTP-inducing
stimulation produced a long-lasting 6-fold increase in the
phosphorylation of synapsin I at its
Ca
2+
/calmodulin-dependent protein kinase
II (CaM kinase II) sites without affecting synapsin I levels. This
effect was fully blocked by either the
N
-methyl-
d
-aspartate receptor antagonist
d
(−)-2-amino-5-phosphonopentanoic acid (APV) or the CaM
kinase II inhibitor KN-62. Our results indicate that LTP expression is
accompanied by persistent changes in presynaptic phosphorylation, and
specifically that presynaptic CaM kinase II activity and synapsin I
phosphorylation may be involved in LTP expression.
Clustered syk tyrosine kinase domains trigger phagocytosis.
Authors: Greenberg S , Chang P , Wang D C , Xavier R , Seed B
Published: July 2002

Phagocytosis is a phylogenetically primitive mechanism adapted by specialized cells of the immune system to ingest particulate pathogens. Recent evidence suggests that the program of specific cytoskeletal rearrangements that underlies phagocytosis may share elements with the antigen receptor signaling pathway in lymphocytes. Tyrosine phosphorylation, necessary for both lymphocyte effector function and phagocytosis, is thought to allow cytoskeletal elements to couple to the intracellular domains of antigen and Fc receptor subunits. We show here that the intracellular domains of the receptors are not inherently required for cytoskeletal coupling. Chimeric transmembrane proteins bearing syk but not src family tyrosine kinase domains are capable of autonomously triggering phagocytosis and redistribution of filamentous actin in COS cells. These responses cannot be initiated by a receptor chimera bearing a point mutation in the syk catalytic domain, and the kinase domain alone is sufficient for initiating cytoskeletal coupling.
The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.
Authors: Tummino P J , Scholten J D , Harvey P J , Holler T P , Maloney L , Gogliotti R , Domagala J , Hupe D
Published: July 2002

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.
Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.
Authors: Berghard A , Buck L B , Liman E R
Published: July 2002

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.
Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.
Authors: Fridell R A , Bogerd H P , Cullen B R
Published: July 2002

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.
Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: Implications for tolerance and immunotherapy
Authors: Wei Chungwen , Willis Richard A. , Tilton Brian R. , Looney R. John , Lord Edith M. , Barth Richard K. , Frelinger John G.
Published: July 2002

Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice. Using a 14-kb genomic DNA region that encompasses the entire human PSA gene and adjacent flanking sequences, we generated a series of human PSA transgenic mice. In the six independent lines of transgenic mice generated, the expression of the human PSA transgene, driven by its owncis-acting regulatory elements, is specifically targeted to the prostate. Tissue distribution analysis demonstrated that PSA transgene expression closely follows the human expression pattern. Immunohistochemical analysis of the prostate tissue also showed that the expression of the PSA transgene is confined to the ductal epithelial cells. Despite expressing PSA as a self-antigen in the prostate, these transgenic mice were able to mount a cytotoxic immune response against PSA expressed by tumor cells, indicating that expression of the transgene has not resulted in complete nonresponsiveness. This transgenic mouse model will provide a well defined system to gain an insight into the mechanisms of nonresponsiveness to PSA, ultimately leading to strategies for immunotherapy of human prostate cancer using PSA as the target antigen.
Transgenic A
1
adenosine receptor overexpression increases myocardial resistance to ischemia
Authors: Matherne G. Paul , Linden Joel , Byford Anne M. , Gauthier Naomi S. , Headrick John P.
Published: July 2002

Activation of myocardial A
1
adenosine receptors (A
1
AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific α-myosin heavy chain promoter and rat A
1
AR cDNA. Heart membranes from two transgene positive lines displayed ≈1,000-fold overexpression of A
1
AR (6,574 ± 965 and 10,691 ± 1,002 fmol per mg of protein vs. 8 ± 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min,
P
< 0.05), lower developed tension (1.2 g vs. 1.6 g,
P
< 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 μM 8-(
p
-sulfophenyl) theophylline but was unaffected by addition of 20 nM
N
6
-cyclopentyladenosine, an A
1
AR agonist. Thus A
1
ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A
1
ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A
1
AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A
1
AR number in murine myocardium. Overexpression of A
1
AR affords additional protection. These data support the concept that genetic manipulation of A
1
AR expression may improve myocardial tolerance to ischemia.
Direct delivery of exogenous MHC class I molecule-binding oligopeptides to the endoplasmic reticulum of viable cells
Authors: Day Patricia M. , Yewdell Jonathan W. , Porgador Angel , Germain Ronald N. , Bennink Jack R.
Published: July 2002

After brief incubation of cells with fluorescein-conjugated peptides that bind major histocompatibility complex (MHC) class I molecules, peptides were detected within the endoplasmic reticulum (ER) by microscopy or by binding to radiolabeled class I molecules. ER delivery of a nonfluorescent peptide was demonstrated using a mAb highly specific for the peptide–class I molecule complex. ER localization of peptides: (
i
) required expression of appropriate class I molecules in the ER but not on the cell surface, (
ii
) was diminished by expression of TAP, the MHC-encoded cytosol to ER peptide transporter, and (
iii
) was blocked by pinocytosis inhibitors but not by brefeldin A. These findings demonstrate the existence of a pathway, likely vesicular in nature, that conveys small extracellular substances to the ER without traversing the Golgi complex or the cytosol. This pathway contributes to the loading of exogenous peptides to MHC class I molecules, but its evolutionary significance may lie in other cellular processes, such as maintaining ER homeostasis or signaling by extracellular substances.
DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life
Authors: Martinez Xavier , Brandt Christian , Saddallah Fatiha , Tougne Chantal , Barrios Christy , Wild Fabian , Dougan Gordan , Lambert Paul-Henri , Siegrist Claire-Anne
Published: July 2002

The relative deficiency of T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) responses in early life is associated with an increased susceptibility to infections by intracellular microorganisms. This is likely to reflect a preferential polarization of immature CD4 T cells toward a Th2 rather than a Th1 pattern upon immunization with conventional vaccines. In this report, it is shown that a single immunization within the first week of life with DNA plasmids encoding viral (measles virus hemagglutinin, Sendai virus nucleoprotein) or bacterial (C fragment of tetanus toxin) vaccine antigens can induce adult-like Th1 or mixed Th1/Th2 responses indicated by production of IgG2a vaccine-specific antibodies and preferential secretion of interferon-γ (IFN-γ) compared with interleukin (IL)-5 by antigen-specific T cells, as well as significant CTL responses. However, in spite of this potent Th1-driving capacity, subsequent DNA immunization was not capable of reverting the Th2-biased responses induced after early priming with a recombinant measles canarypox vector. Thus, DNA vaccination represents a novel strategy capable of inducing Th1 or mixed Th1/Th2 and CTL responses in neonates and early life, providing it is performed prior to exposure to Th2-driving conventional vaccine antigens.
mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm
Authors: Kraemer Doris , Blobel Günter
Published: July 2002

We have identified and molecularly characterized a human protein with a
M
r
of 40,880 Da. After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for
mRN
A binding
p
rotein of 41 kDa). Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope. Immunofluorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshwork-like structures throughout the cytoplasm. The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament- or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton. Double immunofluorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork. Immunogold electronmicroscopy confirmed mrnp 41’s cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes. Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and/or in cytoplasmic transport on, or attachment to, the cytoskeleton. Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in
Schizosaccharomyces pombe
, designated Rae1p, or in
Saccharomyces cerevisiae
, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well.
Comparison of structures and energies of CH
5
2+•
with CH
4
+•
and their possible role in superacidic methane activation
Authors: Rasul Golam , Prakash G. K. Surya , Olah George A.
Published: July 2002

Contrary to previous theoretical studies at the UHF/6-31G*
level, the methonium radical dication CH
5
2+
is not a
C
s
symmetrical structure with a 2e—3c bond but a
C
2v
symmetrical structure 1 with two 2e—3c bonds (at the
UHF/6-31G**, UMP2/6-31G**, and UQCISD(T)/6-311G** levels). The
C
s
symmetrical structure is not even a minimum at the
higher level of calculations. The four hydrogen atoms in 1 are bonded
to the carbon atom by two 2e—3c bonds and the fifth hydrogen atom by
a 2e—2c bond. The unpaired electron of 1 is located in a formal
p-orbital (of the sp
2
-hybridized carbon atom) perpendicular
to the plane of the molecule. Hydrogen scrambling in 1 is however
extremely facile, as is in other C
1
cations. It is found
that the protonation of methane to CH
5
+
decreases the
energy for subsequent homolytic cleavage resulting in the exothermic
(24.1 kcal/mol) formation of CH
4
+•
. Subsequent
reaction with neutral methane while reforming CH
5
+
gives the methyl radical enabling reaction with excess methane to
ethane and H
2
. The overall reaction is endothermic by 11.4
kcal/mol, but offers under conditions of oxidative removal of
H
2
an alternative to the more energetic carbocationic
conversion of methane.
Ol-Prx3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes)
Authors: Joly Jean-Stephane , Bourrat Franck , Nguyen Van , Chourrout Daniel
Published: July 2002

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization ofOl-Prx 3(Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996)Proc. Natl. Acad. Sci. USA93, 10691–10696] and with the humanSHOXgene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B.,et al.(1997)Nat. Genet. 16, 54–62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of thepaired-related(PRX) gene family.Ol-Prx 3, as well as thePRXgenes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern ofOl-Prx 3expression in the central nervous system, which differs markedly from the patterns reported for thePRXgenes,Prx 3excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for thePRX 3family of homeobox genes.
The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres
Authors: Monson Ellen K. , de Bruin Derik , Zakian Virginia A.
Published: July 2002

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997)
Genes Dev.
11, 345–357]. Although
cac1
Δ cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual
cac1
Δ cells switch from transcriptionally “off” to transcriptionally “on” more often per cell cycle than wild-type cells. In addition,
cac1
Δ cells were defective for transcriptional silencing near internal tracts of C
1–3
A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription–PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C
1–3
A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the
Escherichia coli dam
methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.
Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail
Authors: Schweizer Anja , Kornfeld Stuart , Rohrer Jack
Published: July 2002

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp
19
→ Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe
18
→ Ala or Phe
13
→ Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp
19
for sorting of the CD-MPR in endosomes, with Phe
18
, Phe
13
, and several neighboring residues contributing to this function. The addition of the Phe
18
-Trp
19
motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe
18
and Trp
19
with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.
Kinesin-73 in the nervous system of
Drosophila
embryos
Authors: Li Hsi-Ping , Liu Zheng-Mei , Nirenberg Marshall
Published: July 2002

Kinesin-73 cDNA was shown to encode a kinesin heavy chain protein that contains an N-terminal motor domain and a long central region that lacks extensive coiled–coils. The amino acid sequence of the motor domain of kinesin-73 protein is most closely related to the motor domains of
Caenorhabditis elegans
unc-104 and mouse KIF1A. The central region of kinesin-73 protein also is related to unc-104 and KIF1A, but the homology is lower than that of the motor domain. The C-terminal region of kinesin-73 protein contains a cytoskeleton associated protein Gly-rich domain, which is a putative microtubule binding site that is present in some cytoskeleton or dynein-associated proteins. Kinesin-73 mRNA was shown by
in situ
hybridization to be maternally expressed and widely distributed in the syncytial blastoderm embryo. However, later in
Drosophila
embryo development, expression of the kinesin-73 gene becomes restricted mostly to the central and peripheral nervous systems.
CD30 induction of human immunodeficiency virus gene transcription is mediated by TRAF2
Authors: Tsitsikov Erdyni N. , Wright Dowain A. , Geha Raif S.
Published: July 2002

CD30 is a member of the tumor necrosis factor receptor (TNFR) superfamily expressed on activated T and B lymphocytes and natural killer cells. Ligation of CD30 was previously shown to induce NF-κB activation and HIV expression in chronically infected T lymphocytes. In this study, we report that two members of the TNFR-associated factor (TRAF) family of proteins, TRAF1 and TRAF2, independently bind to the intracellular domain of CD30 (CD30IC). Transient overexpression of TRAF2, but not TRAF1, induced NF-κB activation and HIV-1-long terminal repeat-driven transcription in the T cell line, KT3. Moreover, dominant negative mutants consisting of the TRAF domain of TRAF1 and TRAF2 inhibited CD30 induction of NF-κB activation and HIV-1 transcription. These results suggest that CD30 ligation may enhance the expression of HIV via TRAF-2-mediated activation of NF-κB.
cDNA cloning and expression of bovine procollagen I N-proteinase: A new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components
Authors: Colige Alain , Li Shi-Wu , Sieron Aleksander L. , Nusgens Betty V. , Prockop Darwin J. , Lapière Charles M.
Published: July 2002

Procollagen N-proteinase (EC3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers–Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor β or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.
TCOF1
gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region
Authors: Wise Carol A. , Chiang Lydia C. , Paznekas William A. , Sharma Mridula , Musy Maurice M. , Ashley Jennifer A. , Lovett Michael , Jabs Ethylin W.
Published: July 2002

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial
TCOF1
cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of
TCOF1
.
TCOF1
encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development.
Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors
Authors: Walfish Paul G. , Yoganathan Thillainathan , Yang Yong-Fan , Hong Heng , Butt Tauseef R. , Stallcup Michael R.
Published: July 2002

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts
in vitro
in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (
Saccharomyces cerevisiae
) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate β-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.
Expression of mutated Nck SH2/SH3 adaptor respecifies mesodermal cell fate in
Xenopus laevis
development
Authors: Tanaka Masamitsu , Lu Wange , Gupta Ruchika , Mayer Bruce J.
Published: July 2002

Nck is a widely expressed SH2/SH3 adaptor protein containing one SH2 and three SH3 domains. Although Nck is assumed to mediate the formation of protein-protein complexes during signaling, little is currently known about its specific function. We have constructed a series of Nck SH3 and SH2 domain mutants, expressed them in
Xenopus laevis
embryos, and monitored injected embryos for developmental abnormalities. This approach allows correlation of developmental phenotypes with the presence or absence of specific Nck protein-binding domains. We show that microinjection of RNA-encoding Nck with an inactivating mutation in the third SH3 domain (NckK229) into dorsal blastomeres of early embryos caused anterior truncation with high frequency, and membrane localization of both the first and second SH3 domains together was sufficient to induce this anterior-truncation phenotype. Molecular marker analysis of explants revealed that the expression of NckK229 ventralized dorsal mesoderm. Lineage tracing experiments demonstrated that the expression of Nck K229 in dorsal blastomeres affected the migratory properties of mesoderm cells in gastrulation and led to the adoption of a more posterior fate. These data suggest that protein(s) that bind the first and second SH3 domains of Nck can affect the response to signals that establish dorso-ventral patterning, and that protein(s) that bind the third SH3 domain antagonize the ventralizing effect of the first two SH3 domains.
Humanizing the yeast telomerase template
Authors: Henning Karla A. , Moskowitz Naomi , Ashlock Melissa A. , Liu Pu P.
Published: July 2002

Saccharomyces cerevisiae
contains an irregular telomere sequence (TG
1–3
)

n

, which differs from the regular repeat (TTAGGG)

n

found at the telomeres of higher organisms including humans. We have modified the entire 16-nt template region of the
S. cerevisiae
telomerase RNA gene (
TLC1
) to produce (TTAGGG)

n

repeats, the human telomere sequence. Haploid yeast strains with the
tlc1-human
allele are viable with no growth retardation and express the humanized gene at a level comparable to wild type. Southern hybridization demonstrates that (TTAGGG)

n

repeats are added onto the yeast chromosome ends in haploid strains with the
tlc1-human
allele, and sequencing of rescued yeast artificial chromosome ends has verified the addition of human telomeric repeats at the molecular level. These data suggest that the irregularity of the yeast telomere sequence is because of the template sequence of the yeast telomerase RNA. Haploid strains with the
tlc1-human
allele will provide an important tool for studying the function of telomerase and its regulation by telomere-binding proteins, and these strains will serve as good hosts for human artificial chromosome assembly and propagation.
CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promotersin vivo
Authors: Wright Kenneth L. , Chin Keh-Chuang , Linhoff Michael , Skinner Cheryl , Brown Jeffrey A. , Boss Jeremy M. , Stark George R. , Ting Jenny P.-Y.
Published: July 2002

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-γ, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed byin vivogenomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-γ-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.
Rpp2, an essential protein subunit of nuclear RNase P, is required for processing of precursor tRNAs and 35S precursor rRNA in
Saccharomyces cerevisiae
Authors: Stolc Viktor , Katz Alexander , Altman Sidney
Published: July 2002

RPP2
, an essential gene that encodes a 15.8-kDa protein subunit of nuclear RNase P, has been identified in the genome of
Saccharomyces cerevisiae
. Rpp2 was detected by sequence similarity with a human protein, Rpp20, which copurifies with human RNase P. Epitope-tagged Rpp2 can be found in association with both RNase P and RNase mitochondrial RNA processing in immunoprecipitates from crude extracts of cells. Depletion of Rpp2 protein
in vivo
causes accumulation of precursor tRNAs with unprocessed introns and 5′ and 3′ termini, and leads to defects in the processing of the 35S precursor rRNA. Rpp2-depleted cells are defective in processing of the 5.8S rRNA. Rpp2 immunoprecipitates cleave both yeast precursor tRNAs and precursor rRNAs accurately at the expected sites and contain the Rpp1 protein orthologue of the human scleroderma autoimmune antigen, Rpp30. These results demonstrate that Rpp2 is a protein subunit of nuclear RNase P that is functionally conserved in eukaryotes from yeast to humans.
A site-specific recombinase is required for competitive root colonization by
Pseudomonas fluorescens
WCS365
Authors: Dekkers Linda C. , Phoelich Claartje C. , van der Fits Leslie , Lugtenberg Ben J. J.
Published: July 2002

A colonization mutant of the efficient root-colonizing biocontrol strain
Pseudomonas fluorescens
WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to
lppL, lysA, dapF, orf235/233, xerC/sss,
and the largely incomplete
orf238
. The transposon insertion in PCL1233 appeared to be present in the
orf235/233
homologue, designated
orf240
. Introduction of a mutation in the
xerC/sss
homologue revealed that the
xerC/sss
gene homologue rather than
orf240
is crucial for colonization.
xerC
in
Escherichia coli
and
sss
in
Pseudomonas aeruginosa
encode proteins that belong to the λ integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the
xerC/sss
homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe–plant interactions.
Desmosomal adhesion inhibits invasive behavior
Authors: Tselepis Chris , Chidgey Martyn , North Alison , Garrod David
Published: July 2002

Recent studies of human disease and transgenic animal experiments have clearly demonstrated the importance of desmosomes in normal tissue architecture. Furthermore, desmosomal components are down-regulated in certain types of carcinomas, suggesting a possible role for desmosomes in suppression of invasion and metastasis. However, there is no functional evidence to support such a hypothesis. To obtain such evidence, we needed to generate desmosomal adhesion in an invasive cell line. We show that expression of multiple desmosomal components (the desmosomal cadherins, desmocollin and desmoglein, and the armadillo protein, plakoglobin) in nonadhesive L929 fibroblasts generates adhesion in aggregation assays. This adhesion is specifically blocked by short peptides corresponding to the putative cell adhesion recognition sites of desmocollin and desmoglein. This result provides an experimental demonstration of the functional importance of the cell adhesion recognition sites of desmocollin and desmoglein and indicates that both desmosomal cadherins are specifically involved in this adhesion. Moreover, whereas parental L929 cells are strongly invasive into collagen gels, we show that invasion is substantially inhibited in cells transfected with desmosomal components. Invasion is restored by treating the transfected cells with anti-adhesion peptides, indicating that desmosomal adhesion specifically blocks invasion in culture. Our results support the suggestion that desmosomes have a role in suppression of tumor spreading.