bluuebunny/crossref_metadata_2025 huggingface dataset:
Mosaic expression of a tyrosinase fusion gene in albino mice yields a heritable striped coat color pattern in transgenic homozygotes.
Authors: Mintz B , Bradl M
Published: May 2006

Genetically albino mouse eggs were injected with an inducible transgene comprising the wild-type tyrosinase (monophenol, L-dopa: oxygen oxidoreductase, EC 1.14.18.1) cDNA and the metallothionein gene promoter in the expectation that the transgene would be expressed to different extents in the various developing pigment cell clones of at least some individuals, thereby leading to patterned coats. This proved to be the case. Five transgenic mice had lightly pigmented patterns of transverse stripes visualizing melanoblast proliferation and migration dorsoventrally on each side. Similar patterns have been seen in genetically mosaic mouse models produced from conjoined blastomeres of different color genotypes and in many naturally patterned genotypes of mice. Four of the transgenics had heritable patterns and autosomal transgene integration. Their homozygous descendants were darker than hemizygotes and transmitted the basic pattern through many generations. Eyes were also pigmented, with clonal patches of melanized cells in the retinal pigment epithelium. The skin was dark due to many pigmented dermal melanocytes, whereas relatively few were in the hair follicles. This "inversion" is attributable to precocious maturation and migratory arrest of many melanoblasts during passage through the dermis en route to the hair bulbs. Patterning in these mice is considered in light of the view, previously proposed, that phenotypically different clones, or phenoclones, may exist in virtually all mammalian cell types and that many genes may be associated with cis-acting control regions causing variations in their expression that are mitotically perpetuated. We point out that mosaic expression has many implications for development as well as neoplasia. In the latter case, the potential for tumor susceptibility may be affected by clonal variation without further gene mutations or deletions. Thus, mice with variegating transgenes can provide molecular access to gene control mechanisms and to their consequences in development and disease.
Identification of the neurofibromatosis type 1 gene product.
Authors: Gutmann D H , Wood D L , Collins F S
Published: May 2006

The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of approximately 250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to the synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.
A regulated RNA binding protein also possesses aconitase activity.
Authors: Kaptain S , Downey W E , Tang C , Philpott C , Haile D , Orloff D G , Harford J B , Rouault T A , Klausner R D
Published: May 2006

A clone for the iron-responsive element (IRE)-binding protein (IRE-BP) has been transfected and expressed in mouse fibroblasts. The IRE-BP gene product binds IREs with high affinity and specificity. Amino acid alignments reveal that the IRE-BP is 30% identical to mitochondrial aconitase. The 18 active site residues of mitochondrial aconitase are identical to those in the IRE-BP, suggesting that the IRE-BP may possess aconitase activity. After purification of native IRE-BP and immunoaffinity purification of transfected and expressed IRE-BP, we demonstrate that the purified IRE-BP has aconitase activity.
The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix.
Authors: Arents G , Burlingame R W , Wang B C , Love W E , Moudrianakis E N
Published: May 2006

The structure of the octameric histone core of the nucleosome has been determined by x-ray crystallography to a resolution of 3.1 A. The histone octamer is a tripartite assembly in which a centrally located (H3-H4)2 tetramer is flanked by two H2A-H2B dimers. It has a complex outer surface; depending on the perspective, the structure appears as a wedge or as a flat disk. The disk represents the planar projection of a left-handed proteinaceous superhelix with approximately 28 A pitch. The diameter of the particle is 65 A and the length is 60 A at its maximum and approximately 10 A at its minimum extension; these dimensions are in agreement with those reported earlier by Klug et al. [Klug, A., Rhodes, D., Smith, J., Finch, J. T. & Thomas, J. O. (1980) Nature (London) 287, 509-516]. The folded histone chains are elongated rather than globular and are assembled in a characteristic "handshake" motif. The individual polypeptides share a common central structural element of the helix-loop-helix type, which we name the histone fold.
Cell type-specific integrin variants with alternative alpha chain cytoplasmic domains.
Authors: Tamura R N , Cooper H M , Collo G , Quaranta V
Published: May 2006

The integrin heterodimers composed of the alpha 6 subunit with the beta 1 or beta 4 subunit (alpha 6 beta 1 and alpha 6 beta 4) are receptors for laminin and basement membrane components, respectively. The alpha 3 beta 1 integrin recognizes laminin, collagen, fibronectin, or epiligrin. We report the identification of structural variants (A and B) of the alpha 6 and alpha 3 subunits, containing distinct cytoplasmic domains. The expression of one cytoplasmic domain or the other, based probably on alternative exon usage, is cell-type dependent. Most transformed cell lines express both alpha 6A and alpha 6B isoforms, as determined by mRNA amplification or antibody immunoprecipitation. In contrast, embryonic fibroblasts express exclusively alpha 6A, and embryonic stem cells express exclusively alpha 6B. In most normal tissues, both alpha 6 isoforms are detectable, but several tissues express either alpha 6A or alpha 6B. The alpha 3B mRNA was amplified from heart and brain, while all other tissues and cell lines tested contained only alpha 3A mRNA. Alternative cytoplasmic domains may provide a means for varying the cellular responses to the ligands of alpha 6 and alpha 3 integrins according to the cell type.
Identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family.
Authors: Xie S C , Low B G , Nagel R J , Kramer K K , Anthony R V , Zoli A P , Beckers J F , Roberts R M
Published: May 2006

Pregnancy in cattle and sheep can be diagnosed by the presence of a conceptus-derived antigen in maternal serum that is secreted by trophoblast and placental tissue primarily as an acidic component of Mr 67,000. Molecular cloning of its cDNA reveals that the antigen belongs to the aspartic proteinase family and has greater than 50% amino acid sequence identity to pepsin, cathepsin D, and cathepsin E. The inferred sequences of the ovine and bovine polypeptides show approximately 73% identity to each other. Critical amino acid substitutions at the active site regions suggest that both proteins are enzymatically inactive. The antigen is a product of trophoblast binucleate cells that invade maternal endometrium at implantation sites.
Molecular phylogeny of the superorder Archonta.
Authors: Adkins R M , Honeycutt R L
Published: May 2006

The superorder Archonta has been hypothesized to include primates, tree shrews, bats, and flying lemurs as descendants of a common ancestor. More recently, a diphyletic origin for bats has been proposed. To evaluate these hypotheses, the nucleotide sequence of the mitochondrial cytochrome oxidase subunit II gene was determined from a bushbaby (Galago senegalensis), flying lemur (Cynocephalus variegatus), tree shrew (Tupaia glis), spear-nosed bat (Phyllostomus hastatus), rousette bat (Rousettus leschenaulti), and nine-banded armadillo (Dasypus novemcinctus) and was compared with published sequences of a human, cow, and mouse. Phylogenetic analyses of the sequences give evidence that primates, tree shrews, and flying lemurs have a recent common ancestor but that bats are genealogically distant. The monophyletic origin of bats is supported. Contrary to interpretations based on morphological data, tree shrews are shown to be no more closely affiliated with primates than are flying lemurs. Analyses of the cytochrome oxidase subunit II gene give marginally more support to a Dermoptera-Scandentia clade than to a Dermoptera-Primates clade.
The concept of symmorphosis: a testable hypothesis of structure-function relationship.
Authors: Weibel E R , Taylor C R , Hoppeler H
Published: May 2006

The hypothesis that, in biological organisms, structural design is matched to functional demand is difficult to test because it is largely based on anecdotal evidence suggesting economic design. The hypothesis of symmorphosis postulates a quantitative match of design and function parameters within a defined functional system; because of its stringency it is refutable and can, therefore, be subjected to empirical test, for example, by assessing whether the structures that support the pathway for oxygen from the lung to the consumer in muscle cells are quantitatively adjusted to the limit of functional performance of the respiratory system. The study of allometric and adaptive variation leads to the conclusion that the hypothesis of symmorphosis is acceptable for all internal compartments of the respiratory system (blood, heart, muscle capillaries, and mitochondria), whereas it must be refuted for the lung that forms the interface to the environment.
A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.
Authors: Neuhaus J M , Sticher L , Meins F , Boller T
Published: May 2006

Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.
A steroid-inducible gene expression system for plant cells.
Authors: Schena M , Lloyd A M , Davis R W
Published: May 2006

Promoters that allow the selective induction of gene expression in vivo constitute an important methodology in eukaryotic organisms such as yeast and the fruit fly, but to date no such system has been described for higher plants. Given the fact that mammalian steroid receptors can function as hormone-dependent inducers of gene expression in heterologous systems, the feasibility of using mammalian steroid hormones as selective inducers of plant gene expression was investigated. Here it is shown that the glucocorticoid receptor expressed in plant cells is capable of activating a test gene linked to glucocorticoid response elements, providing the transfected plant cells are treated with glucocorticoid hormone. Nanomolar concentrations of glucocorticoids are sufficient to induce gene expression more than 150-fold, without causing detectable alterations in the physiology of the cultured plant cells. These findings indicate that glucocorticoid induction of steroid-responsive promoters should provide a general method for regulating gene expression in plant cells and imply that such a system might ultimately function in whole plants such as Arabidopsis thaliana.
Elevated content of the tyrosine kinase substrate phospholipase C-gamma 1 in primary human breast carcinomas.
Authors: Arteaga C L , Johnson M D , Todderud G , Coffey R J , Carpenter G , Page D L
Published: May 2006

Phospholipase C-gamma 1 (PLC-gamma 1) is a substrate for several receptor tyrosine kinases and its catalytic activity is increased by tyrosine phosphorylation. However, the biological significance of this molecule in normal or malignant human epithelial cell proliferation is unknown. We determined the relative content of PLC-gamma 1 in primary human mammary carcinomas and in nonmalignant mammary tissues. By Western blot and immunohistochemistry, considerably higher levels of PLC-gamma 1 protein were detectable in the majority of carcinomas and in one of two benign fibroadenomas compared to normal breast tissues. In 18 of 21 carcinomas that contained high levels of PLC-gamma 1, the presence of phosphotyrosine on PLC-gamma 1 could also be detected. All carcinomas in which tyrosine phosphorylated PLC-gamma 1 was present also expressed detectable levels of the epidermal growth factor receptor or erbB-2, two tyrosine kinases known to phosphorylate this enzyme. Thus, a high percentage of mammary carcinomas concomitantly display increased levels of receptor tyrosine kinases and a direct tyrosine phosphorylation substrate, thereby potentially amplifying two successive steps in a signal transduction pathway.
Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei.
Authors: Volloch V , Schweitzer B , Zhang X , Rits S
Published: May 2006

A substantial amount of cytochrome oxidase subunit III (COIII) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand COIII RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand COIII RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited COIII RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of COIII RNA. The presence of negative-strand complements of COIII RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.
A protein kinase is present in a complex with adenovirus E1A proteins.
Authors: Kleinberger T , Shenk T
Published: May 2006

A kinase activity can be immunoprecipitated in a complex that includes adenovirus E1A proteins. In vitro, this activity phosphorylated other E1A-associated proteins, as well as added E1A and histone H1 proteins. The E1A-associated kinase activity was cleared from extracts with an antibody to cyclin A, but not with antibody to cyclin B. The formation of a complex that included the kinase activity required amino acids 30-60 and 122-129 on the E1A proteins, sequences needed for association of E1A proteins with cyclin A and the retinoblastoma protein and implicated in control of cell growth. The complex of E1A-associated proteins included a 33-kDa ATP-binding protein, similar in size to a cyclin A-associated cdc2 kinase family member. Sucrose gradient analysis revealed two distinct E1A-containing complexes with the kinase activity. We suggest that E1A proteins may affect cellular proliferation by interacting with a member of the cdc2 kinase family and thereby influencing its activity.
Low-resolution genome map of the malaria mosquito Anopheles gambiae.
Authors: Zheng L B , Saunders R D , Fortini D , della Torre A , Coluzzi M , Glover D M , Kafatos F C
Published: May 2006

We have microdissected divisions of the Anopheles gambiae polytene chromosomes, digested the DNAs with a restriction enzyme, and PCR-amplified the DNA fragments to generate a set of pooled probes, each corresponding to approximately 2% of the mosquito genome. These divisional probes were shown to have high complexity. Except for those derived from near the centromeres, they hybridize specifically with their chromosomal sites of origin. Thus, they can be used to map cloned DNAs by a dot blot procedure, which is much more convenient than in situ hybridization to polytene chromosomes. We discuss additional potential uses of these probes, such as easier isolation of molecular markers and genes, including those that cross-hybridize with clones available from other insects. It is expected that the probes will substantially accelerate molecular genetic analysis of this most important malaria vector.
Inactivation of the cholinesterase gene by Alu insertion: possible mechanism for human gene transposition.
Authors: Muratani K , Hada T , Yamamoto Y , Kaneko T , Shigeto Y , Ohue T , Furuyama J , Higashino K
Published: May 2006

The human cholinesterase (ChE) gene from a patient with acholinesterasemia was cloned and analyzed. By using ChE cDNA as a probe, four independent clones were isolated from a genomic library constructed from the patient's DNA. Sequencing analysis of all of the four clones revealed that exon 2 of the ChE gene was disrupted by a 342-base-pair (bp) insertion of Alu element, including a poly(A) tract of 38 bp, which showed 93% sequence homology with a current type of human Alu consensus sequence. Southern blot analysis showed that the Alu insertion occurred in both alleles of the patient and was inherited in the patient's family. This Alu insertion was flanked by 15-bp of target site duplication in exon 2 corresponding to positions 1062-1076 of ChE cDNA, indicating that an Alu element could have been integrated by retrotransposition. Thus, this case provides an important clue to the mechanism of inactivation of a gene by integration of a retrotransposon.
Premature termination of transcription from the P1 promoter of the mouse c-myc gene.
Authors: Wright S , Mirels L F , Calayag M C , Bishop J M
Published: May 2006

Modulation of transcriptional elongation within the c-myc gene is thought to play a major role in determining levels of c-myc mRNA in both normal and tumor cells. A discrete site of blockage to transcriptional elongation has previously been localized at the 3' end of exon 1 of the mouse and human c-myc genes. We here identify an additional site of transcriptional attenuation that is located between the P1 and P2 promoters of the c-myc gene and that mediates premature termination of transcripts initiating from the P1 promoter. A 95-nucleotide DNA fragment derived from this region prematurely terminated transcription when placed downstream from the promoter of the H-2Kbm1 gene and assayed by expression in Xenopus oocytes. We also show that the previously identified attenuation signal in exon 1 of the mouse c-myc gene can mediate premature termination of P1-initiated transcripts. Premature termination of P1-initiated transcripts presumably increases transcription from the downstream P2 promoter; aberrant regulation of this termination may explain the increased use of the P1 promoter that is characteristic of certain tumors in which myc is overexpressed.
High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons.
Authors: McKendry R , John J , Flavell D , Müller M , Kerr I M , Stark G R
Published: May 2006

2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive mutants were isolated previously at a low frequency (ca. 10(-8)) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-alpha. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, greater than 3 x 10(-7). Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-alpha have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U3 and U4 are unresponsive to IFN-alpha and, surprisingly, also unresponsive to IFN-gamma. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and double-stranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U3A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (alpha/beta) IFNs. Band-shift complementation assays show that U3A contains the E gamma subunit but does not contain an active E alpha subunit after treatment with IFN-alpha.
Localization and regulation of c-fos and c-jun protooncogene induction by systolic wall stress in normal and hypertrophied rat hearts.
Authors: Schunkert H , Jahn L , Izumo S , Apstein C S , Lorell B H
Published: May 2006

The effect of changes in left ventricular (LV) systolic force generation on cardiac c-fos and c-jun protooncogene expression was studied by using isolated beating hearts from male Wistar rats. An isovolumic buffer-perfused heart preparation was utilized in which coronary flow and heart rate were held constant and increments in LV balloon volume were used to generate defined levels of LV systolic wall stress. Using Northern and slot-blot analyses, we found that LV tissue from control hearts that generated high levels of LV systolic wall stress expressed 3- to 4.4-fold higher c-fos and c-jun mRNA levels in comparison with tissue from the respective flaccid right ventricles, and in comparison with LV tissue from hearts that generated minimal LV systolic wall stress. To distinguish the role of passive LV diastolic wall stretch from active LV force generation, we found that distension of the LV balloon per se did not have a significant effect on protooncogene induction in hearts perfused with 2,3-butanedione monoxime, which prevents systolic cross-bridge cycling and force generation. In additional hearts studied at a constant LV balloon volume to generate an LV end-diastolic pressure of 10 mm Hg, c-fos mRNA levels were proportional to the magnitude of peak LV systolic wall stress (r = 0.823, P less than 0.05). In these protocols, Fos protein was localized by immunohistochemistry in myocyte nuclei with minimal staining in fibroblasts and vascular smooth muscle. When c-fos and c-jun mRNA expression was compared in hearts with chronic LV hypertrophy due to ascending aortic banding and age-matched control hearts that generated similar incremental levels of LV systolic wall stress, significantly lower levels of c-fos and c-jun mRNA were measured in the hypertrophied hearts. However, there was no difference in protooncogene mRNA expression in response to stimulation by the Ca2+ ionophore A23187. These data suggest that, in this isolated isovolumic beating heart preparation, the active generation of an acute increment in LV systolic force independent of passive diastolic myocardial stretch causes a rapid induction of both c-fos and c-jun, which is down-regulated in the presence of established LV hypertrophy.
Rapid induction of the c-jun protooncogene in the avian oviduct by the antiestrogen tamoxifen.
Authors: Lau C K , Subramaniam M , Rasmussen K , Spelsberg T C
Published: May 2006

This report describes a rapid regulation of the expression of the c-jun protooncogene by the antiestrogen tamoxifen (Tam). The c-jun protooncogene codes for an important component of the AP-1 transcription factor complex, which regulates the expression of many unlinked genes. Repeated experiments have shown that Tam rapidly increases the steady-state c-jun mRNA levels in the avian oviduct but decreases the levels in the liver. The Tam effects are time- and dose-dependent. These results are supported by other studies that have demonstrated that 17 beta-estradiol decreases steady-state levels of c-jun protooncogene mRNA in oviducts of animals fully withdrawn from estradiol. The effect of Tam in the avian oviduct is in contrast to the reported effects of Tam on the expression of practically all other genes in the avian oviduct and other animal tissues. Transcription analyses using nuclear runoff experiments with oviduct nuclei demonstrate a decrease in the c-jun gene transcription within minutes after Tam treatment with a return to 75% of control values by 4 hr. The fact that Tam transiently decreases the transcription of the c-jun gene but increases the steady-state c-jun mRNA levels suggests that Tam must alter both transcriptional and post-transcriptional events. The results support a role of the c-jun protooncogene as a regulatory gene in the cascade model for steroid action whereby steroids rapidly regulate the regulatory genes, which in turn regulate many other structural genes.
Molecular localization of the t(11;22)(q24;q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization.
Authors: Selleri L , Hermanson G G , Eubanks J H , Lewis K A , Evans G A
Published: May 2006

Chromosome translocations are associated with a variety of human leukemias, lymphomas, and solid tumors. To localize molecular markers flanking the t(11;22) (q24;q12) breakpoint that occurs in virtually all cases of Ewing sarcoma and peripheral neuroepithelioma, high-resolution chromosomal in situ suppression hybridization was carried out using a panel of cosmid clones localized and ordered on chromosome 11q. The location of the Ewing sarcoma translocation breakpoint was determined relative to the nearest two cosmid markers on 11q, clones 23.2 and 5.8, through the analysis of metaphase chromosome hybridization. By in situ hybridization to interphase nuclei, the approximate physical separation of these two markers was determined. In both Ewing sarcoma and peripheral neuroepithelioma, cosmid clone 5.8 is translocated from chromosome 11q24 to the derivative chromosome 22 and a portion of chromosome 22q12 carrying the leukemia inhibitory factor gene is translocated to the derivative chromosome 11. The physical distance between the flanking cosmid markers on chromosome 11 was determined to be in the range of 1000 kilobases, and genomic analysis using pulsed-field gel electrophoresis showed no abnormalities over a region of 650 kilobases in the vicinity of the leukemia inhibitory factor gene on chromosome 22. This approach localizes the Ewing sarcoma breakpoint to a small region on chromosome 11q24 and provides a rapid and precise technique for the molecular characterization of chromosomal aberrations.
Dystrophin is transcribed in brain from a distant upstream promoter.
Authors: Boyce F M , Beggs A H , Feener C , Kunkel L M
Published: May 2006

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is expressed in brain as well as muscle. The role of dystrophin in the brain is not clear, though one-third of Duchenne muscular dystrophy patients exhibit some degree of mental retardation. We have isolated the genomic region encoding the alternative 5' terminus of dystrophin used in the brain. Primer extension and polymerase chain reaction assays on RNA demonstrate that this region contains an alternative promoter for dystrophin used in the brain. Physical mapping of this region indicates that this brain promoter is located greater than 90 kilobases 5' to the promoter used in muscle and 400 kilobases from exon 2 to which it is spliced. The large physical distance between the promoters, taken together with their known tissue selectivities, suggests that in certain patients a deletion of either dystrophin promoter might give rise to reduced dystrophin expression selective to brain or muscle. We have identified one such individual with specific deletion of the dystrophin muscle promoter, giving rise to Becker muscular dystrophy, and we predict that specific loss of the brain promoter may be one cause of X chromosome-linked mental retardation.
Cocaine-induced reduction of brain neuropeptide Y synthesis dependent on medial prefrontal cortex.
Authors: Wahlestedt C , Karoum F , Jaskiw G , Wyatt R J , Larhammar D , Ekman R , Reis D J
Published: May 2006

Repeated administration of cocaine elicits substantial, long-lasting, but reversible reductions in neuropeptide Y (NPY) and NPY mRNA in the rat cerebral cortex and nucleus accumbens. The NPY reduction appears to be mediated through a decrease in NPY biosynthesis, occurring transneuronally, perhaps in response to changes in synaptic dopamine associated with mesolimbic and mesocortical dopamine neurons. The medial prefrontal cortex appears necessary for maintenance of cocaine's action on this neuronal network since excitotoxic lesions of this area prevented (lesion before cocaine) and reversed (lesion after cocaine) the reductions in NPY elicited by the cocaine. NPY may be a sensitive marker for chronic cocaine use. Its decrease may relate to the anxiety and depression associated with cocaine withdrawal in humans.
Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination.
Authors: Eid J , Sollner-Webb B
Published: May 2006

A calmodulin-neomycin-resistance fusion gene was introduced into Trypanosoma brucei by electroporation, and stably transformed cell lines were obtained. In all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin DNA. This unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants. It should now be possible to systematically manipulate the trypanosome genome, directing predetermined mutations to virtually any chromosomal locus.
An angiogenin-binding protein from endothelial cells.
Authors: Hu G F , Chang S I , Riordan J F , Vallee B L
Published: May 2006

A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. Binding of 125I-labeled bovine angiogenin (125I-Ang) to AngBP occurs with an apparent Kd approximately 5 x 10(-10) M and is specific, saturable, and inhibited by excess unlabeled angiogenin. 125I-Ang can be crosslinked efficiently to AngBP by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. Bovine ribonuclease A competes with the binding of 125I-Ang to AngBP, but lysozyme does not. Direct binding to AngBP of 125I-labeled bovine ribonuclease A is, however, much weaker than that of 125I-Ang. Two enzymatically active derivatives of angiogenin cleaved at residues 60-61 and 67-68, respectively, fail to induce angiogenesis and also bind to AngBP only weakly. AngBP has been isolated by treatment of cells with heparan sulfate, affinity chromatography on angiogenin-Sepharose of the material dissociated from the cell surface, and gel filtration HPLC. The results suggest that AngBP has the characteristics of a receptor that may likely function in angiogenesis.
Splice junctions follow a 205-base ladder.
Authors: Beckmann J S , Trifonov E N
Published: May 2006

Of all aspects of mRNA maturation the accuracy of intervening sequence excision and exon ligation is, perhaps, the most enigmatic. Attempts to identify the essential elements involved in this process have thus far not yielded any satisfactory answer as to what structural (sequence) features are prerequisite for the vital precision of this process. In our search for underlying structural orders we asked whether exons and introns had any positional preferences within a gene. This analysis led to the unexpected discovery that the DNA length is synchronized between successive 3' splicing sites as well as between successive 5' splicing sites, with a frame of approximately 205 base pairs. This observation reveals additional organization of genes in eukaryotes and, perhaps, links gene splicing with chromatin structure.
Protein dynamics in the bacteriorhodopsin photocycle: submillisecond Fourier transform infrared spectra of the L, M, and N photointermediates.
Authors: Braiman M S , Bousché O , Rothschild K J
Published: May 2006

The usefulness of stroboscopic time-resolved Fourier transform IR spectroscopy for studying the dynamics of biological systems is demonstrated. By using this technique, we have obtained broadband IR absorbance difference spectra after photolysis of bacteriorhodospin with a time resolution of approximately 50 microseconds, spectral resolution of 4 cm-1, and a detection limit of delta A less than or equal to 10(-4). These capabilities permit observation of detailed structural changes in individual residues as bacteriorhodopsin passes through its L, M, and N intermediate states near physiological temperatures. When combined with band assignments based on isotope labeling and site-directed mutagenesis, the stroboscopic Fourier transform IR difference spectra show that on the time scale of the L intermediate, Asp-96 has an altered environment that may be accompanied by change in its protonation state. On the time scale of the L----M transition, this Asp-96 perturbation/deprotonation is largely reversed, and Asp-85 becomes protonated. During the M----N transition, Asp-85 appears to remain protonated but undergoes a change in its environment as evidenced by a shift of vC = O from 1761 to 1755 cm-1. The retention of a proton on Asp-85 in the N state indicates that the proton transferred from the Schiff base to this residue in the L----M step is not released to the extracellular medium during the same photocycle, but rather during a subsequent one. Also during the M----N transition, Asp-96 undergoes a deprotonation (possibly for the second time in a single photocycle). Bands in the amide I and amide II spectral regions in the M----N difference spectrum indicate the occurrence of a conformational change involving one or more peptide groups in the protein backbone.
Intracellular distribution of microinjected antisense oligonucleotides.
Authors: Leonetti J P , Mechti N , Degols G , Gagnor C , Lebleu B
Published: May 2006

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.
Malaria parasite chitinase and penetration of the mosquito peritrophic membrane.
Authors: Huber M , Cabib E , Miller L H
Published: May 2006

Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-279]. In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 3.2.1.14), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane.
Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils.
Authors: Seder R A , Paul W E , Dvorak A M , Sharkis S J , Kagey-Sobotka A , Niv Y , Finkelman F D , Barbieri S A , Galli S J , Plaut M
Published: May 2006

Splenic and bone marrow cells from normal mice, and from mice that have been polyclonally activated by injection of anti-IgD antibody, contain cells that produce interleukin 4 (IL-4) in response to crosslinkage of Fc epsilon receptors (Fc epsilon R) or Fc gamma R or to ionomycin. Isolated Fc epsilon R+ cells have recently been shown to contain all of the IL-4-producing capacity of the nonlymphoid compartment of spleen and bone marrow. Here, purified Fc epsilon R+ cells are shown to be enriched in cells that contain histamine and express alcian blue-positive cytoplasmic granules. By electron microscopy, the vast majority of cytoplasmic granule-containing cells are basophils; they constitute approximately 25% and approximately 50%, respectively, of Fc epsilon R+ spleen and bone marrow cells from anti-IgD-injected mice. The Fc epsilon R- populations contain cells that form colonies typical of mast cells. The Fc epsilon R+ populations also contain cells that, upon culture with IL-3, form colonies of alcian blue-positive cells, but (in contrast to colonies derived from Fc epsilon R- populations) the colonies are small, and all the cells die within 2-3 weeks. The Fc epsilon R+ cells synthesize histamine during a 60-hr culture with IL-3, while the Fc epsilon R- cells do not. These results indicate that IL-4-producing Fc epsilon R+ cells are highly enriched in basophils.
Sperm attraction to a follicular factor(s) correlates with human egg fertilizability.
Authors: Ralt D , Goldenberg M , Fetterolf P , Thompson D , Dor J , Mashiach S , Garbers D L , Eisenbach M
Published: May 2006

Spermatozoa normally encounter the egg at the fertilization site (in the Fallopian tube) within 24 hr after ovulation. A considerable fraction of the spermatozoa ejaculated into the female reproductive tract of mammals remains motionless in storage sites until ovulation, when the spermatozoa resume maximal motility and reach the fertilization site within minutes. The nature of the signal for sperm movement is not known, but one possible mechanism is attraction of spermatozoa to a factor(s) released from the egg. We have obtained evidence in favor of such a possibility by showing that human spermatozoa accumulate in follicular fluid in vitro. This accumulation into follicular fluid was higher by 30-260% than that observed with buffer alone and was highly significant (P less than 10(-8)). Not all of the follicular fluids caused sperm accumulation; however, there was a remarkably strong correlation (P less than 0.0001) between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized. These findings suggest that attraction may be a key event in the fertilization process and may give an insight into the mechanism underlying early egg-sperm communication.
Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.
Authors: Colicelli J , Nicolette C , Birchmeier C , Rodgers L , Riggs M , Wigler M
Published: May 2006

Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.
Aspartylglycosaminuria in the Finnish population: identification of two point mutations in the heavy chain of glycoasparaginase.
Authors: Mononen I , Heisterkamp N , Kaartinen V , Williams J C , Yates J R , Griffin P R , Hood L E , Groffen J
Published: May 2006

Aspartylglycosaminuria is an inherited lysosomal storage disease caused by deficiency of glycoasparaginase (EC 3.5.1.26) and occurs with higher frequency among Finns than other populations. We have purified human glycoasparaginase and determined about 90% of the amino acid sequence of its light subunit and greater than 70% of that of its heavy subunit by Edman degradation and mass spectrometry. Additional sequence data were obtained from the cloning and subsequent nucleotide analysis of a cDNA corresponding to the normal human glycoasparaginase gene. The enzyme is encoded by a single mRNA as a single polypeptide that is posttranslationally processed to generate the subunits and is glycosylated. After preparing first-strand cDNA from leukocyte and fibroblast total RNA, we used the polymerase chain reaction to amplify the glycoasparaginase cDNA of eight Finnish aspartylglycosaminuria patients. We demonstrate that the Finnish patients' mRNA sequence differed from the normal sequence by two single-base changes six nucleotides apart from one another in the heavy chain of glycoasparaginase. The first change resulted in the replacement of arginine by glutamine (R161Q), whereas the second change resulted in a cysteine to serine substitution (C163S). Both mutations resulted in novel restriction endonuclease sites and were present in all eight Finnish aspartylglycosaminuria patients originating from different pedigrees, but they were absent from Finnish and non-Finnish controls and a non-Finnish case of aspartylglycosaminuria. These results indicate molecular homogeneity in aspartylglycosaminuria alleles in the Finnish population.
Transcription mediated by a cAMP-responsive promoter element is reduced upon activation of dopamine D2 receptors.
Authors: Montmayeur J P , Borrelli E
Published: May 2006

Dopaminergic D2 receptors mediate the effect of dopamine on cellular effector systems by means of guanine nucleotide-binding proteins (G proteins). The major biochemical effect evoked by these receptors is the inhibition of adenylyl cyclase. As a consequence, the activation of D2 receptors lowers the intracellular cAMP level. Two cDNAs, originated by alternative splicing of the same gene, have been isolated: D2A and D2B. They code for two proteins of 444 and 415 amino acids. These proteins display high affinity for selective D2 dopamine ligands. D2A differs from D2B by an insertion of 87 nucleotides in its cDNA, which is located in a region of the protein considered important for the coupling to G proteins. To investigate functional differences between the two dopamine D2 receptor isoforms, we transiently expressed them in cultured cells. To do so we developed an assay to study membrane receptors that are coupled to the adenylyl cyclase. Using this assay, we were able to show that the stimulation of the adenylyl cyclase induced by the activation of the beta 2-adrenergic receptor is inhibited more efficiently by D2B than D2A. The effects elicited by the D2 receptors are mediated by pertussis toxin-sensitive G proteins. Treatment of transfected cells with pertussis toxin abolishes the inhibitory effects in a dose- and receptor isoform-dependent manner. Our results suggest that the two dopamine receptor isoforms are differentially coupled to G proteins.
Proteolysis of the class II-associated invariant chain generates a peptide binding site in intracellular HLA-DR molecules.
Authors: Roche P A , Cresswell P
Published: May 2006

HLA-DR molecules are heterodimeric transmembrane glycoproteins that associate intracellularly with a polypeptide known as the invariant (I) chain. Shortly before expression of the HLA-DR alpha beta dimer on the cell surface, however, the I chain is removed from the intracellular alpha beta I complex by a mechanism thought to involve proteolysis. In this report, we show that treatment of purified alpha beta I with the cysteine proteinase cathepsin B results in the specific proteolysis of the HLA-DR-associated I chain in vitro. As a consequence of this, the I chain is removed and free alpha beta dimers are released from alpha beta I. Although alpha beta I fails to bind an immunogenic peptide, the released alpha beta dimers acquire the ability to bind the peptide after proteolysis of the I chain. These results suggest that the I chain inhibits immunogenic peptide binding to alpha beta I early during intracellular transport and demonstrate that proteolysis is likely to be the in vivo mechanism of I chain removal.
Vanadate-resistant yeast mutants are defective in protein glycosylation.
Authors: Ballou L , Hitzeman R A , Lewis M S , Ballou C E
Published: May 2006

Spontaneous recessive orthovanadate-resistant mutants of Saccharomyces cerevisiae were obtained in five complementation groups, and all show defects in protein glycosylation that mimic the previously isolated mnn mutants. Three of the groups are allelic to the known mnn8, mnn9, and mnn10 mutants, whereas the other two groups show other glycosylation defects. The vanadate-resistant phenotype was associated with enhanced hygromycin B sensitivity. The glycosylation phenotypes of the mutants are all reflections of defects in glycoprotein trafficking, and the easy isolation of vanadate-resistant or hygromycin B-sensitive mutants should facilitate the study of this process.