bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.
Authors: Xiang M , Gan L , Zhou L , Klein W H , Nathans J
Published: July 2002

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.
Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.
Authors: Smith J L , Silveira L A , Spudich J A
Published: July 2002

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.
Characterization and developmental patterns of telomerase expression in plants
Authors: Fitzgerald Matthew S. , McKnight Thomas D. , Shippen Dorothy E.
Published: July 2002

Telomerase activity is developmentally regulated in mammals. Here
we examine telomerase activity in plants, whose development differs in
fundamental ways from that of animals. Using a modified version of the
telomere repeat amplification protocol (TRAP) assay, we detected an
activity in extracts from carrots, cauliflower, soybean,
Arabidopsis
, and rice with all the characteristics
expected for a telomerase synthesizing the plant telomere repeat
sequence TTTAGGG. The activity was dependent on RNA and protein
components, required dGTP, dATP, and dTTP, but not dCTP, and generated
products with a seven nucleotide periodicity. Telomerase activity was
abundant in cauliflower meristematic tissue and undifferentiated cells
from
Arabidopsis
, soybean, and carrot suspension
cultures, but was low or not detectable in a sampling of differentiated
tissues from mature plants. Telomerase from cauliflower meristematic
tissues exhibited relaxed DNA sequence requirements, which might
reflect the capacity to form telomeres on broken chromosomes
in
vivo
. The dramatic differences in telomerase expression and
their correlation with cellular proliferation capacity mirror changes
in human telomerase levels during differentiation and immortalization.
Hence, telomerase activation appears to be a conserved mechanism
involved in conferring long-term proliferation capacity.
Frequency of migrants and migratory activity are genetically correlated in a bird population: Evolutionary implications
Authors: Pulido Francisco , Berthold Peter , van Noordwijk Arie J.
Published: July 2002

Most migratory bird populations are composed of individuals that migrate and individuals that remain resident. While the role of ecological factors in maintaining this behavioral dimorphism has received much attention, the importance of genetic constraints on the evolution of avian migration has not yet been considered. Drawing on the recorded migratory activities of 775 blackcaps (Sylvia atricapilla) from a partially migratory population in southern France, we tested two alternative genetic models about the relationship between incidence and amount of migratory activity. The amount of migratory activity could be the continuous variable “underlying” the phenotypic expression of migratory urge, or, alternatively, the expression of both traits could be controlled by two separate genetic systems. The distributions of migratory activities in five different cohorts and the inheritance pattern derived from selective breeding experiments both indicate that incidence and amount of migratory activity are two aspects of one trait. Thus, all birds without measurable activity have activity levels at the low end of a continuous distribution, below the limit of expression or detection. The phenotypic dichotomy “migrant–nonmigrant” is caused by a threshold which may not be fixed but influenced both genetically and environmentally. This finding has profound implications for the evolution of migration: the transition from migratoriness to residency should not only be driven by selection favoring resident birds but also by selection for lower migratory activity. This potential for selection on two aspects, residency and migration distance, of the same trait may enable extremely rapid evolutionary changes to occur in migratory behavior.
Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.
Authors: Wersto R P , Rosenthal E R , Crystal R G , Spring K R
Published: July 2002

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.
Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.
Authors: Bourinet E , Soong T W , Stea A , Snutch T P
Published: July 2002

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.
Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.
Authors: Batista F D , Efremov D G , Burrone O R
Published: July 2002

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.
Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family.
Authors: Gehrig P M , Krzyzaniak A , Barciszewski J , Biemann K
Published: July 2002

The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond.
Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope HNK-1
Authors: Terayama Koji , Oka Shogo , Seiki Takashi , Miki Yukari , Nakamura Akemi , Kozutsumi Yasunori , Takio Koji , Kawasaki Toshisuke
Published: July 2002

The HNK-1 carbohydrate epitope is characteristically expressed on a series of cell adhesion molecules and also on some glycolipids in the nervous system over a wide range of species from insect to mammal. The HNK-1 epitope is involved in cell–cell and/or cell–substrate interaction and recognition during the development of the nervous system. In this study, we isolated a novel glucuronyltransferase from rat brain, which is a key enzyme of the biosynthesis of the HNK-1 epitope on glycoproteins. Based on the partial amino acid sequences, we isolated cDNA encoding the glucuronyltransferase. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein with 347 amino acids and had no detectable similarity with any other proteins of known functions, including glucuronyltransferases of the liver and olfactory epithelium. Expression of a soluble recombinant form of the enzyme in COS-1 cells produced an active glucuronyltransferase. The selective expression of the glucuronyltransferase gene in the nervous system was consistent with the almost exclusive localization of the HNK-1 epitope in the nervous system. Transfection of the glucuronyltransferase cDNA into COS-1 cells induced not only expression of the HNK-1 epitope on the cell surface but also marked morphological changes of the cells, suggesting that the HNK-1 epitope associates with the cell–substratum interaction.
Drosophila drop-dead
mutations accelerate the time course of age-related markers
Authors: Rogina Blanka , Benzer Seymour , Helfand Stephen L.
Published: July 2002

Mutations of the
drop-dead
gene in
Drosophila melanogaster
lead to striking early death of the adult animal. At different times, after emergence from the pupa, individual flies begin to stagger and, shortly thereafter, die. Anatomical examination reveals gross neuropathological lesions in the brain. The life span of flies mutant for the
drop-dead
gene is four to five times shorter than for normal adults. That raises the question whether loss of the normal gene product might set into motion a series of events typical of the normal aging process. We used molecular markers, the expression patterns of which, in normal flies, change with age in a manner that correlates with life span. In the
drop-dead
mutant, there is an acceleration in the temporal pattern of expression of these age-related markers.
Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus
Authors: Martínez Miguel A. , Verdaguer Nuria , Mateu Mauricio G. , Domingo Esteban
Published: July 2002

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature.
Heat transduction in rat sensory neurons by calcium-dependent activation of a cation channel
Authors: Reichling David B. , Levine Jon D.
Published: July 2002

The mechanism of heat transduction in vertebrate sensory neurons was investigated
in vitro
by using cultured dorsal root ganglion neurons from adult rat. In response to a physiologically relevant range of stimulus temperatures (23–45°C), a subpopulation of small dorsal root ganglion neurons are depolarized by a cation current (heat-activated current,
I
heat
) that is antagonized by extracellular cesium. Heat-induced single-channel currents in cell-attached patches are evoked at a similar range of temperatures.
I
heat
is a calcium-dependent current activated indirectly by heat-evoked release of calcium from intracellular stores. This suggests that the channel itself is not the transducer of thermal energy. Similar to nociceptive heat sensation
in vivo
,
I
heat
is enhanced by the hyperalgesic agent prostaglandin E
2
and only partially adapts during prolonged heat stimuli. To our knowledge, these data provide the first demonstration that ion channels can mediate heat transduction in mammalian sensory neurons and provide evidence that heat causes the channels to open via an increase in the intracellular second messenger calcium.
RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box
Authors: Forget Diane , Robert François , Grondin Gilles , Burton Zachary F. , Greenblatt Jack , Coulombe Benoit
Published: July 2002

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription
in vitro
. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.
Can increasing carbon dioxide cause climate change?
Authors: Lindzen Richard S.
Published: July 2002

The realistic physical functioning of the greenhouse effect is
reviewed, and the role of dynamic transport and water vapor is
identified. Model errors and uncertainties are quantitatively compared
with the forcing due to doubling CO
2
, and they are shown to
be too large for reliable model evaluations of climate sensitivities.
The possibility of directly measuring climate sensitivity is reviewed.
A direct approach using satellite data to relate changes in globally
averaged radiative flux changes at the top of the atmosphere to
naturally occurring changes in global mean temperature is described.
Indirect approaches to evaluating climate sensitivity involving the
response to volcanic eruptions and Eocene climate change are also
described. Finally, it is explained how, in principle, a climate that
is insensitive to gross radiative forcing as produced by doubling
CO
2
might still be able to undergo major changes of the
sort associated with ice ages and equable climates.
SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor α (RARα) and PLZF-RARα oncoproteins associated with acute promyelocytic leukemia
Authors: Hong Suk-Hyun , David Gregory , Wong Chi-Wai , Dejean Anne , Privalsky Martin L.
Published: July 2002

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARα. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARα and PLZF-RARα oncoproteins retain the ability of RARα to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors.
Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate
Authors: Verrier Florence C. , Charneau Pierre , Altmeyer Ralf , Laurent Stephanie , Borman Andrew M. , Girard Marc
Published: July 2002

The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+CCR-5+HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1BX08isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1LAIstrain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.
Strained DNA is kinked by low concentrations of Zn2+
Authors: Han Wenhai , Dlakic Mensur , Zhu Yinwen Judy , Lindsay S. M. , Harrington Rodney E.
Published: July 2002

A novel atomic force microscope with a magnetically oscillated tip has provided unprecedented resolution of small DNA fragments spontaneously adsorbed to mica and imagedin situin the presence of divalent ions. Kinks (localized bends of average angle 78°) were observed in axially strained minicircles consisting of tandemly repeated d(A)5and d(GGGCC[C]) sequences. The frequency of kinks in identical minicircles increased 4-fold in the presence of 1 mM Zn2+compared with 1 mM Mg2+. Kinking persisted in mixed Mg2+/Zn2+electrolytes until the Zn2+concentration dropped below 100 μM, indicating that this type of kinking may occur under physiological conditions. Kinking appears to replace intrinsic bending, and statistical analysis shows that kinks are not localized within any single sequence element. A surprisingly small free energy is associated with kink formation.
The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole
Authors: Springer Mark S. , Burk Angela , Kavanagh John R. , Waddell Victor G. , Stanhope Michael J.
Published: July 2002

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.
Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice
Authors: Wang Bing-yan , El-Badri Nagwa S. , Good Cherry, and Robert A.
Published: July 2002

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.
Floral homeotic genes were recruited from homologous MADS-box genes preexisting in the common ancestor of ferns and seed plants
Authors: Münster Thomas , Pahnke Jens , Di Rosa Alexandra , Kim Jan T. , Martin William , Saedler Heinz , Theissen Günter
Published: July 2002

Flowers
sensu lato
are short, specialized axes bearing closely aggregated sporophylls. They are typical for seed plants (spermatophytes) and are prominent in flowering plants
sensu stricto
(angiosperms), where they often comprise an attractive perianth. There is evidence that spermatophytes evolved from gymnosperm-like plants with a fern-like mode of reproduction called progymnosperms. It seems plausible, therefore, that the stamens/carpels and pollen sacs/nucelli of spermatophytes are homologous to fern sporophylls and sporangia, respectively. However, the exact mode and molecular basis of early seed and flower evolution is not yet known. Comparing flower developmental control genes to their homologs from lower plants that do not flower may help to clarify the issue. We have isolated and characterized MADS-box genes expressed in gametophytes and sporophytes of the fern
Ceratopteris
. The data indicate that at least two different MADS-box genes homologous to floral homeotic genes existed in the last common ancestor of contemporary vascular plants, some descendants of which underwent multiple duplications and diversifications and were recruited into novel developmental networks during the evolution of floral organs.
Fas-induced proteolytic activation and intracellular redistribution of the stress-signaling kinase MEKK1
Authors: Deak Joseph C. , Cross Janet V. , Lewis Margaret , Qian Yongyi , Parrott Louis A. , Distelhorst Clark W. , Templeton Dennis J.
Published: July 2002

The stress-activated protein kinase (SAPK, alternatively JNK) is activated rapidly by cell stress stimuli such as inflammatory cytokines and oxidative stress, and more slowly by the initiation of the apoptotic cell death response by events such as ligation of the Fas protein. Mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1) is an activator of SAPK, serving as a SAPK-kinase-kinase through intermediate phosphorylation of the SAPK kinase SEK1. By sequencing proteolytic cleavage products of MEKK1, we found that the proapoptotic protease caspase 3 (CPP32) cleaves MEKK1 after residue D68 both
in vivo
and
in vitro
. Cleavage of MEKK1 after D68 is blocked by viral and chemical protease inhibitors. Cleavage of MEKK1 at D68 changes the intracellular distribution of the protein from a Triton-insoluble compartment to a Triton-soluble compartment, reflected in a redistribution from a particulate to a diffuse cytoplasmic staining seen by immunofluorescence. Activation of both SAPK and MEKK1 after Fas ligation is prevented by both viral and chemical caspase 3 inhibitors, which in contrast fail to block activation of SAPK by rapidly acting cell stresses. Stress factor-induced SAPK signaling is not dependent on caspase 3 function. We propose that two mechanisms of stress signaling through MEKK1 exist. One is rapid, independent of proteases, and occurs in the particulate Triton-insoluble compartment. The other is more slowly activated and involves liberation of particulate MEKK1 by proteolytic cleavage and activation by caspase 3.
Increased E2F1 activity induces skin tumors in mice heterozygous and nullizygous for p53
Authors: Pierce Angela M. , Gimenez-Conti Irma B. , Schneider-Broussard Robin , Martinez Luis A. , Conti Claudio J. , Johnson David G.
Published: July 2002

The p16
INK4a
-cyclin D–retinoblastoma tumor suppressor pathway is disrupted in most human cancers, and it has been suggested that the subsequent release of E2F transcription factors from inhibitory complexes may be a key event in tumor development. We described recently the generation of transgenic mice with E2F1 gene expression targeted to squamous epithelial tissues by a keratin 5 (K5) promoter. In the present study, K5 E2F1 transgenic mice were crossed with p53 null mice to examine functional interactions between E2F1 and p53
in vivo.
We find that E2F1-induced apoptosis of epidermal keratinocytes is reduced in K5 E2F1 transgenic mice lacking p53, whereas E2F1-induced hyperproliferation is unaffected by p53 status. We also find that K5 E2F1 transgenic mice heterozygous or nullizygous for p53 develop spontaneous skin carcinomas, which normally are rare in p53-deficient mice. The timing of tumor development correlates with the level of E2F1 transgene expression and the status of p53. In primary transgenic keratinocytes, the major change in E2F1 DNA-binding activity is the generation of a complex also containing the retinoblastoma tumor suppressor protein. Nevertheless, the expression and associated kinase activity of cyclin E, a known target for E2F transcriptional activity, is elevated significantly in K5 E2F1 transgenic keratinocytes. These findings firmly establish that increased E2F1 expression can contribute to tumor development and suggest that p53 plays an important role in eliminating cells with deregulated E2F1 activity.
A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the
cog/cog
mouse: A model of human endoplasmic reticulum storage diseases
Authors: Kim Paul S. , Hossain Shaikh Abu , Park Young-Nam , Lee Ike , Yoo Sung-Eun , Arvan Peter
Published: July 2002

Newly synthesized thyroglobulin (Tg), the major secretory glycoprotein of the thyroid gland, folds and homodimerizes in the endoplasmic reticulum (ER) before its export to the site of iodination, where it serves as the precursor for thyroid hormone synthesis. In families with defective Tg export, affected individuals suffer from a thyroidal ER storage disease characterized by a distended thyrocyte ER containing misfolded Tg, along with induced ER molecular chaperones. Inherited as an autosomal recessive trait, deficient Tg causes congenital hypothyroidism in newborns that, if untreated, results in goiter along with serious cognitive and growth defects. Recently, a similar phenotype has been observed in inbred
cog/cog
mice, although the precise molecular defect has remained undefined. Here, we have isolated and cloned a full-length 8.5-kb Tg cDNA from
cog/cog
mice and unaffected isogenic AKR/J mice. Comparison of the complete sequences reveals that
cog/cog
mice express a Leu-2263 → Pro missense mutation in the acetylcholinesterase-homology domain of Tg. Heterologous expression studies in COS cells indicate that
cog
Tg exhibits a severe defect in exit from the ER. Site-directed mutagenesis of
cog
Tg to convert the single amino acid back to Leu-2263 restores normal Tg secretion. We conclude that the
cog
mutation in Tg is responsible for this ER storage disease that causes thyroid dyshormonogenesis.
Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: Structure and stability of the
per
-thiaproline mutant of annexin V
Authors: Budisa Nediljko , Minks Caroline , Medrano F. Javier , Lutz Jürgen , Huber Robert , Moroder Luis
Published: July 2002

Residue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic acid [thiaproline, Pro(S)], a non-natural amino acid analog of proline, into human recombinant annexin V was achieved with a proline-auxotrophic
Escherichia coli
strain by fermentation procedures in minimal medium. Quantitative replacement of proline with thiaproline was confirmed by mass-spectrometric, amino acid, and x-ray crystallographic analyses. The wild-type protein and its
per
-Pro(S) mutant were found to crystallize isomorphously and to show identical three-dimensional structures in crystals. In solution the dichroic properties of the wild-type and
per
-Pro(S) protein confirmed nearly identical overall folds. From thermal denaturation experiments, however, a reduced
T
m
(−4.5 K) value was determined whereas the van’t Hoff enthalpy and entropy were not significantly affected. Therefore, protein mutants containing bioactive amino acid analogs like thiaproline at multiple sites would be expected to fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.
The relationships between notochord and floor plate in vertebrate development revisited
Authors: Teillet M.-A. , Lapointe F. , Le Douarin N. M.
Published: July 2002

By using the quail–chicken chimera system, we have previously shown that during development of the spinal cord, floor plate cells are inserted between neural progenitors giving rise to the alar plates. These cells are derived from the regressing Hensen’s node or cordoneural hinge (HN-CNH). This common population of HN-CNH cells gives rise to three types of midline descendants: notochord, floor plate, and dorsal endoderm. Here we find that
HNF3
β, an important gene in the development of the midline structures, is continuously expressed in the HN-CNH cells and their derivatives, floor plate, notochord, and dorsal endoderm. Experiments in which the notochord was removed in the posterior region of either normal chicken or of quail–chicken chimeras in which a quail HN had been grafted showed that the floor plate develops in a cell-autonomous manner in the absence of notochord. Absence of floor plate observed at the posterior level of the excision results from removal of HN-CNH material, including the future floor plate, and not from the lack of an inductive signal of notochord origin.
Reduction in levels of the cyclin-dependent kinase inhibitor p27
kip-1
coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells
Authors: Dao Mo A. , Taylor Naomi , Nolta Jan A.
Published: July 2002

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15
INK4B
blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27
kip-1
blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15
INK4B
and increased colony formation and retroviral-mediated transduction of primary human CD34
+
cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27
kip-1
coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.
Chromosome-specific molecular organization of maize (
Zea mays
L.) centromeric regions
Authors: Ananiev E. V. , Phillips R. L. , Rines H. W.
Published: July 2002

A set of oat–maize chromosome addition lines with individual maize (
Zea mays
L.) chromosomes present in plants with a complete oat (
Avena sativa
L.) chromosome complement provides a unique opportunity to analyze the organization of centromeric regions of each maize chromosome. A DNA sequence, MCS1a, described previously as a maize centromere-associated sequence, was used as a probe to isolate cosmid clones from a genomic library made of DNA purified from a maize chromosome 9 addition line. Analysis of six cosmid clones containing centromeric DNA segments revealed a complex organization. The MCS1a sequence was found to comprise a portion of the long terminal repeats of a retrotransposon-like repeated element, termed CentA. Two of the six cosmid clones contained regions composed of a newly identified family of tandem repeats, termed CentC. Copies of CentA and tandem arrays of CentC are interspersed with other repetitive elements, including the previously identified maize retroelements Huck and Prem2. Fluorescence
in situ
hybridization revealed that CentC and CentA elements are limited to the centromeric region of each maize chromosome. The retroelements Huck and Prem2 are dispersed along all maize chromosomes, although Huck elements are present in an increased concentration around centromeric regions. Significant variation in the size of the blocks of CentC and in the copy number of CentA elements, as well as restriction fragment length variations were detected within the centromeric region of each maize chromosome studied. The different proportions and arrangements of these elements and likely others provide each centromeric region with a unique overall structure.
Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in
Arabidopsis
allopolyploids
Authors: Chen Z. Jeffrey , Comai Luca , Pikaard Craig S.
Published: July 2002

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural
Arabidopsis suecica
, an allotetraploid (amphidiploid) hybrid of
Arabidopsis thaliana
and
Cardaminopsis arenosa
, the
A. thaliana
rRNA genes are repressed. Interestingly,
A.
thaliana
rRNA gene silencing is variable in synthetic
Arabidopsis
suecica
F
1
hybrids. Two generations are needed for
A. thaliana
rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic
A. suecica
to tetraploid
A. thaliana
yielded progeny with active
A. thaliana
rRNA genes and, in some cases, silenced
C. arenosa
rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that
A. thaliana
and
C. arenosa
rRNA genes can be expressed transiently in the other species. Transfected
A. thaliana
genes are also active in
A. suecica
protoplasts in which chromosomal
A. thaliana
genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.
Social transmission of reproductive behavior increases frequency of inherited disorders in a young-expanding population
Authors: Austerlitz Frédéric , Heyer Evelyne
Published: July 2002

The observation of high frequencies of certain inherited disorders in the population of Saguenay–Lac Saint Jean can be explained in terms of the variance and the correlation of effective family size (EFS) from one generation to the next. We have shown this effect by using the branching process approach with real demographic data. When variance of EFS is included in the model, despite its profound effect on mutant allele frequency, any mutant introduced in the population never reaches the known carrier frequencies (between 0.035 and 0.05). It is only when the EFS correlation between generations is introduced into the model that we can explain the rise of the mutant alleles. This correlation is described by a
c
parameter that reflects the dependency of children’s EFS on their parents’ EFS. The
c
parameter can be considered to reflect social transmission of demographic behavior. We show that such social transmission dramatically reduces the effective population size. This could explain particular distributions in allele frequencies and unusually high frequency of certain inherited disorders in some human populations.
Two functionally dependent acetylcholine subunits are encoded in a single
Caenorhabditis elegans
operon
Authors: Treinin Millet , Gillo Boaz , Liebman Lilach , Chalfie Martin
Published: July 2002

The
deg-3
gene from the nematode
Caenorhabditis elegans
encodes an α subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele,
u662
, which produced neuronal degeneration. Because
deg-3
cDNAs contain the SL2 trans-spliced leader, we suggested that
deg-3
was transcribed as part of a
C. elegans
operon. Here we show that
des-2
, a gene in which mutations suppress
deg-3(u662)
, is the upstream gene in that operon. The
des-2
gene also encodes an α subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (
i
)
des-2
is needed for the
deg-3(u662)
degenerations
in vivo
; (
ii
) an acetylcholine-gated channel is formed in
Xenopus
oocytes when both subunits are expressed but not when either is expressed alone; and (
iii
) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a
u662
-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.
Structure of the elongating ribosome: Arrangement of the two tRNAs before and after translocation
Authors: Nierhaus Knud H. , Wadzack Jörg , Burkhardt Nils , Jünemann Ralf , Meerwinck Wolf , Willumeit Regine , Stuhrmann Heinrich B.
Published: July 2002

The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins. The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques. However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time. Here we analyze a pair of ribosomal complexes being either in the pre- or post-translocational states that represent the main states of the elongating ribosome. Both complexes were derived from one preparation. The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the
Escherichia coli
ribosome and the tRNA structure. The mass center of gravity of the (tRNA)
2
mRNA complex moves within the ribosome by 12 ± 4 Å in the course of translocation as previously reported. The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110° ± 10° before and after translocation. The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18°.