bluuebunny/crossref_metadata_2025 huggingface dataset:
Does biased gene conversion influence polymorphism in the circumsporozoite protein-encoding gene of Plasmodium vivax?
Authors: Arnot D E , Barnwell J W , Stewart M J
Published: May 2006

Variation between North Korean and Latin American isolates in the circumsporozoite (CS) protein encoding gene of the human malaria parasite Plasmodium vivax was studied. Polymorphic positions are confined to the central tandemly repeated sequences. Nucleotide substitutions in the tandem repeats produce variants; these substituted positions within the repeat array tend to be conserved between genes. The North Korean CS gene has a short insertion after the repeats encoding a 4-amino acid repeat (Ala-Gly-Gly-Asn) not found in the New World P. vivax genes. This sequence is found both flanking and within the tandem repeats of the CS genes of several strains of the Southeast Asian simian malaria parasite, Plasmodium cynomolgi. The intraspecific conservation of positions of variants within tandem repeat arrays and the interspecific conservation of probably ancestral repeat motifs at the end of these arrays are consistent with the occurrence of nonreciprocal genetic exchanges between the tandem repeats of these genes. However, a striking asymmetry in strand nucleotide composition within the tandem repeats of all CS genes leads us to suggest that biased correction of heteroduplexes formed during recombination plays a role in the evolution of these genes.
A spontaneously opened ring chromosome of Drosophila melanogaster has acquired He-T DNA sequences at both new telomeres.
Authors: Traverse K L , Pardue M L
Published: May 2006

Ring chromosomes that have been opened to give linear chromosomes offer an opportunity to study the DNA sequences associated with new chromosome ends. The Drosophila melanogaster chromosome C(1)A was originally a ring chromosome, consisting of two linked X chromosomes, and thus had no telomeres. This chromosome has spontaneously opened in polytene region 13, a region near the middle of the euchromatic arm of the X chromosome. The opening of the ring has produced two new telomeres on the C(1)A chromosome. Each of the new telomeres has acquired He-T DNA sequences. He-T DNA is a complex family of repeated sequences found in the telomeric and pericentric heterochromatin of D. melanogaster chromosomes. He-T DNA sequences are detected, at various levels, in the most distal band on the end of each polytene chromosome in all D. melanogaster stocks. To our knowledge, these sequences have never been detected within the euchromatic chromosomal regions in any stock. The strong correlation between He-T DNA sequences and telomeric regions suggests that He-T sequences may have a role in organizing or maintaining the ends of chromosomes. The association of He-T DNA with newly acquired telomeres in a formerly euchromatic region, polytene region 13, strengthens this correlation.
Chromosomal location of three spectrin genes: relationship to the inherited hemolytic anemias of mouse and man.
Authors: Birkenmeier C S , McFarland-Starr E C , Barker J E
Published: May 2006

Three genetic loci in the mouse affect the synthesis and assembly of the erythrocyte membrane skeleton. The spherocytosis and jaundiced loci affect the membrane skeletal protein known as spectrin. The normoblastosis locus affects the spectrin binding protein called ankyrin. We have obtained genetic data that define the linkage relationships among three spectrin genes and the spherocytosis and jaundiced loci. The erythroid alpha-spectrin gene is tightly linked to the spherocytosis locus on chromosome 1 and the jaundiced locus is on chromosome 12, tightly linked to the erythroid beta-spectrin gene. The brain alpha-spectrin (alpha-fodrin) gene is located on the centromeric end of chromosome 2 and is not closely linked to any previously mapped erythroid or neurological mutation. These results are consistent with the hypothesis that defects in the alpha- and beta-spectrin genes cause the spherocytosis and jaundiced hemolytic anemias in mice. All five loci studied are located within chromosomal segments that are conserved between mouse and man. Analysis of the data from the chromosome 12 study defines a new order for the genes on that chromosome and delineates the largest mouse/human conserved chromosomal segment yet known.
Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair.
Authors: Schaaper R M
Published: May 2006

To investigate the mechanisms of spontaneous mutation in the Escherichia coli mutD5 mutator strain, 502 mutations generated in this strain in the N-terminal part of the lacI gene were sequenced (i-d mutations). Since the mutator strength of this strain depends on the medium in which it grows, mutations were analyzed in both minimal medium (moderate mutator activity) and rich medium (high mutator activity). In either case, 95% of all mutations were base substitutions and 5% were single-base deletions. However, the nature and site distribution of the base substitutions differed dramatically for the two conditions. In minimal medium (mutation frequency, 480-fold above background), a majority (62%) were transversions, notably A.T----T.A at three 5'-GTGG-3' sequences. Most (64%) of the transitions under this condition occurred at specific sequences that are suggestive of a "dislocation" type of mutagenesis. In rich medium (mutation frequency, 37,000-fold above background), 90% of the base substitutions were transitions. These observations suggest that different modes of mutagenesis operate under the two conditions. mutD5 cells have been reported to be defective in exonucleolytic proofreading during DNA replication. The present data suggest that mutD cells in rich medium also suffer a defect in mutHLS-encoded mismatch correction. This hypothesis was confirmed by the direct measurement of mismatch repair in mutD5 cells by transfection of M13mp2 heteroduplex DNA.
Conserved upstream sequences of human class II major histocompatibility genes enhance expression of class II genes in wild-type but not mutant B-cell lines.
Authors: Sloan J H , Boss J M
Published: May 2006

Class II major histocompatibility genes contain a conserved upstream sequence (CUS) that is important in the expression of these genes. This region has been divided into two major elements, the X box and the Y box. The ability of these elements to mediate transcription of a heterologous promoter was assayed upon transfection into a B-cell line (Raji), a class II-specific trans-acting factor-deficient B-cell line (RJ2.2.5 cells), and a T-cell line (Jurkat). The results showed that the X box element was responsible for directing tissue-specific expression when Raji cells were compared to Jurkat cells. The X box could not direct expression of the heterologous promoter in the trans-acting factor-deficient cell line, indicating that the X box is an ultimate target of the missing or defective factor in the RJ2.2.5 cell line. The Y box directed an equal but extremely low level of transcription in this system in both the mutant and wild-type B-cell lines, suggesting that this element is not involved in B-cell expression or as a target of the mutant factor.
Cancer antigens are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid cell line.
Authors: Shiraishi Y , Soma H
Published: May 2006

We have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the "panning" method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes, der(14)t(14;14) (p11;q11),t(6;?)(p25;?) and t(9;?)(q34;?), besides t(17;?) (q25;?) found in the OVC-antigen-positive clones and t(5;?) (p13;?),t(7;?)(q36;?) found in the ML-antigen-positive clones. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer.
Trans-activation of the human immunodeficiency virus long terminal repeat by the hepatitis B virus X protein.
Authors: Seto E , Yen T S , Peterlin B M , Ou J H
Published: May 2006

Human immunodeficiency virus 1 has been implicated as the main etiologic agent of the acquired immunodeficiency syndrome. However, other infectious agents may accelerate the progression of this disease. In particular, hepatitis B virus has been suggested as one such cofactor. Therefore, we have investigated the effects of hepatitis B virus gene products on expression of the human immunodeficiency virus I in transient transfection studies of Jurkat lymphoblastic T cells, using as reporter the chloramphenicol acetyltransferase gene coupled to the long terminal repeat of human immunodeficiency virus I. As measured by the amount of chloramphenicol acetyltransferase activity, gene expression directed by the human immunodeficiency virus I long terminal repeat increased approximately 10-fold in response to the hepatitis B virus X protein. This trans-activation by the X protein is multiplicative with the effect of phorbol esters and can be accounted for by an increase in the steady-state level of chloramphenicol acetyltransferase mRNA. Analysis of deletion and clustered point mutants in the long terminal repeat indicated that the X protein exerts its effect through multiple cis-acting sites. These results provide a possible molecular basis for the association of hepatitis B virus and the acquired immunodeficiency syndrome and confirm that the X protein is a transcriptional transactivator.
Cloning and substrate specificity of a human phenol UDP-glucuronosyltransferase expressed in COS-7 cells.
Authors: Harding D , Fournel-Gigleux S , Jackson M R , Burchell B
Published: May 2006

A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDP-glucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector lambda gt11. The 2.4-kilobase cDNA contained an open reading frame of 1593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of approximately 55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of approximately 3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase.
Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.
Authors: Gorelick R J , Henderson L E , Hanser J P , Rein A
Published: May 2006

All retroviruses encode a nucleic acid-binding or nucleocapsid protein that is believed to be associated with RNA in the virion. Further, all retroviral nucleocapsid proteins contain either one or two copies of the sequence Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys. The conservation of this sequence suggested that it is important for virus replication, and its resemblance to the "zinc-finger" sequences found in eukaryotic transcription factors raised the possibility that it recognizes specific sequences in viral RNA during retrovirus assembly. We used oligonucleotide-directed mutagenesis to generate a series of mutations in the nucleocapsid protein-coding region of Moloney murine leukemia virus. These mutations changed single amino acids, including each of the cysteines, to serine. The mutant viral genomes direct the synthesis of virus particles; these particles lack detectable viral RNA but do contain significant levels of cellular RNAs. Thus it appears that the mutations have destroyed the ability of the viral proteins to specifically package viral RNA during virus assembly. We propose that the conserved sequence in retroviral nucleocapsid proteins functions in RNA sequence recognition and suggest that it is evolutionarily related to the zinc fingers that recognize specific sequences in double-stranded DNA.
Infrared spectroelectrochemistry of bacteriochlorophylls and bacteriopheophytins: Implications for the binding of the pigments in the reaction center from photosynthetic bacteria
Authors: Mäntele W. G. , Wollenweber A. M. , Nabedryk E. , Breton J.
Published: May 2006

The IR spectra of the bacteriochlorophyll a and b cations and the bacteriopheophytin a and b anions were obtained by using an IR and optically transparent electrochemical cell. Prominent effects of radical formation on the vibrational spectra were found for bands assigned to the ester, keto, and acetyl C=O groups and for vibrations from macrocycle bonds. The (radical-minus-neutral) difference spectra are compared to the light-induced difference spectra of the primary donor photooxidation and the intermediary acceptor photoreduction in the reaction center of photosynthetic bacteria. Light-induced absorbance changes from bacteriochlorophyll a-containing reaction centers bear striking similarities to the electrochemically induced absorbance changes observed upon formation of bacteriochlorophyll a
+
in vitro
. Comparison of the radical formation
in vitro
in a hydrogen-bonding or a nonhydrogen-bonding solvent suggests an ester C=O group hydrogen bonded in the neutral state but free in the cation state. For the keto C=O group, the same comparison indicates one free carbonyl group. The (anion-minus-neutral) difference spectra of bacteriopheophytin a and b exhibit a single band in the ester C=O frequency range. In contrast, two bands are observed in the difference spectra of the intermediary acceptor reduction in the reaction center of
Rhodopseudomonas viridis
. The higher frequency band exhibits a sensitivity to
1
H-
2
H exchange, which suggests a contribution from a protonated carboxyl group of an amino acid side chain.
Rapid activation of protein kinase C in isolated rat liver nuclei by prolactin, a known hepatic mitogen.
Authors: Buckley A R , Crowe P D , Russell D H
Published: May 2006

Rat liver nuclei pure by enzymatic and electron microscope criteria contain protein kinase C (PKC) that can be activated several hundredfold within 3 min of addition of prolactin or phorbol 12-tetradecanoate 13-acetate. Rat prolactin stimulated PKC maximally at 10(-12) M, whereas ovine prolactin was maximally stimulatory at 10(-10) M. Activation was time and dose dependent, exhibited a biphasic pattern, and was blocked by anti-prolactin antiserum, by PKC inhibitors such as 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and sphingosine, and by cyclosporine. Moreover, the ability of prolactin to activate nuclear PKC was inhibited totally by a monoclonal antibody to the rat liver prolactin receptor, implicating a prolactin receptor-mediated activation process. Epidermal growth factor (EGF), a liver mitogen, caused a lesser but significant activation of nuclear PKC. However, EGF and suboptimal prolactin were synergistic. Human growth hormone, which has lactogenic properties, stimulated PKC activity, whereas nonlactogenic substances such as ovine growth hormone, insulin, dexamethasone, and 8-bromo-cAMP were inactive. That this may be a general mechanism for prolactin is suggested by the ability of prolactin to stimulate PKC 140-fold in rat splenocyte nuclei. Prolactin has comitogenic properties in lymphocytes.
Oligomeric structure of a prototype retrovirus glycoprotein.
Authors: Einfeld D , Hunter E
Published: May 2006

The structure of the Rous sarcoma virus envelope glycoprotein complex was studied by sedimentation gradient centrifugation analyses of detergent-solubilized wild-type and mutant envelope (env) gene products. These studies show that the envelope glycoprotein forms an oligomer during biosynthesis, which is most likely a trimer, and that this is the form of the complex found in virions. Our results are consistent with oligomer formation and transport out of the endoplasmic reticulum being closely linked. From analyses of mutant envelope proteins we conclude that the extracellular domain of the glycoprotein is sufficient for oligomer formation but that the transmembrane domain is required to stabilize this complex. Additional experiments suggest that interactions between external domains of the membrane-spanning, gp37 polypeptides are those most important for the formation of trimers. The significance of these observations to retroviral replication and implications for antiviral drug development are discussed.
Stimulation of arachidonic acid release and inhibition of mitogenesis by cloned genes for muscarinic receptor subtypes stably expressed in A9 L cells.
Authors: Conklin B R , Brann M R , Buckley N J , Ma A L , Bonner T I , Axelrod J
Published: May 2006

A family of genes encoding four distinct muscarinic receptors (designated m1-m4) has been cloned and stably expressed in A9 L cells. When the m1 and m3 receptors were stimulated with carbachol, there was a rapid rise of liberated arachidonic acid, inositol phosphates, and cAMP, while m2 and m4 receptor stimulation had no detectable stimulation of these second messengers. Pretreatment with phorbol 12-myristate 13-acetate (PMA) caused a marked acceleration and amplification of m1 and m3 receptor-mediated arachidonic acid release. In contrast, m1- and m3-mediated inositol phosphate formation was inhibited by the same PMA pretreatment. Arachidonic acid release was unaffected by manipulations of cAMP levels. Arachidonic acid production was inhibited by calcium-free medium and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8; an inhibitor of cytosolic calcium mobilization) yet was unaffected by verapamil, a calcium-channel blocker. These experiments show that arachidonic acid release induced by the m1 and m3 receptors is regulated independently of phospholipase C and cAMP accumulation. Carbachol stimulation of the m1 and m3 cAMP accumulation. Carbachol stimulation of the m1 and m3 receptors also markedly decreased mitogenesis as measured by thymidine incorporation. The m1 receptor-mediated inhibition of mitogenesis could be partially blocked by indomethacin, a cyclooxygenase inhibitor. The inhibition of mitogenesis could be mimicked by cAMP elevation.
Control of Torpedo acetylcholine receptor biosynthesis in Xenopus oocytes.
Authors: Buller A L , White M M
Published: May 2006

The nicotinic acetylcholine receptor (AcChoR) of Torpedo electroplax is a multisubunit transmembrane glycoprotein complex with a subunit stoichiometry of alpha 2 beta gamma delta. The RNAs for the separate subunits were transcribed in vitro from cDNAs inserted in pSP64T vectors and microinjected in Xenopus oocytes. Microinjected in vitro-transcribed RNAs were stable, with a half-life of 72 hr. Xenopus oocytes assembled functional AcChoRs from the subunit-specific RNAs. These receptors were inserted in the cell membrane and could be detected as early as 6 hr after RNA microinjection. The oocyte-expressed AcChoR subunits could be immunoprecipitated with anti-Torpedo AcChoR subunit antibodies. Expression of the AcChoR in oocytes proceeded linearly for 72 hr after microinjection. While the amount of RNA injected did not alter the linearity of the expression time course, the rate of receptor expression in oocytes showed a saturable dependence on RNA concentration. Varying the relative amount of alpha-subunit RNA microinjected into oocytes had a striking effect on receptor expression. This effect was specific for the alpha-subunit. These results suggest that transcript availability may control receptor expression in Xenopus oocytes. In addition, the availability of the alpha-subunit may be a limiting factor for receptor expression.
Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning.
Authors: Alvarez-Buylla A , Theelen M , Nottebohm F
Published: May 2006

The higher vocal center (HVc) of the canary brain projects to two forebrain nuclei: robustus archistriatalis (RA) and area X of lobus parolfactorius. The time of birth of HVc neurons projecting to these two regions was determined by combining [3H]thymidine autoradiography and retrograde fluorogold uptake. Birds were sacrificed at 13 months of age, 4 days after fluorogold injections into area X or RA. A single injection of [3H]thymidine in ovo (embryonic day 9) labeled 76% of area X-projecting cells and 0.8% of cells projecting to RA. The great majority of RA-projecting cells were produced during posthatching development (posthatching day 10-240; P10-P240), with a peak at P60 and a hiatus at P120. HVc reaches full adult size by P240, yet at that age the production of new RA-projecting cells continued at a rate comparable to that recorded during posthatching development. Late production of neurons interconnecting two distant regions of the brain may regulate source to target cell population size. Male canaries start to sing at P40. During subsequent months, they imitate external models and their song becomes more structured and stereotyped. At sexual maturity (P240), song is stable. Three interpretations are offered: (i) neurogenesis of RA-projecting cells is related to learning, and learning continues even after achievement of pattern stability; (ii) neurogenesis of RA-projecting cells is not related to learning; (iii) the production of RA-projecting cells serves different purposes during development and after sexual maturity.
Determination of DNA structures by NMR and distance geometry techniques: a computer simulation.
Authors: Pardi A , Hare D R , Wang C
Published: May 2006

Computer simulations have been performed to determine how accurately and precisely structures of DNA oligomers can be generated from distance data obtained from two-dimensional NMR experiments. A hexamer fragment d(CGAATT) of the Dickerson dodecamer [Drew, H.R., Wing, R.M., Takano, T., Broka, C., Tanaha, S., Itakura, K. & Dickerson, R.E. (1981) Proc. Natl. Acad. Sci. USA 78, 2179-2183] was used as the model structure in these simulations. Protons were added to the coordinates of the original x-ray structure, which was then subjected to a regularization procedure to minimize deviations from standard bond lengths and bond angles. The proton-proton distances normally observed in NMR experiments were measured from this regularized target structure and used as input for a distance geometry algorithm. Distance geometry structures were generated from two distance sets, one with essentially exact distances (+/- 0.005 A) and one set with a precision (+/- 0.2 A) that simulates an optimal NMR experiment. The results of these calculations were used to judge how accurately and precisely the following helical parameters could be reproduced from this simulated NMR distance data: helical twist, helical rise, dislocation, roll, tilt, glycosidic angle, delta torsion angle, and pseudorotation angle. These data provide a basis from which to judge the quality of DNA structures produced from real NMR experiments.
Reduction of polygalacturonase activity in tomato fruit by antisense RNA
Authors: Sheehy Raymond E. , Kramer Matthew , Hiatt William R.
Published: May 2006

Polygalacturonase [PG; poly(1,4-α-D-galacturonide) glycanhydrolase; EC 3.2.1.15] is expressed in tomato only during the ripening stage of fruit development. PG becomes abundant during ripening and has a major role in cell wall degradation and fruit softening. Tomato plants were transformed to produce antisense RNA from a gene construct containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in reverse orientation. The construct was integrated into the tomato genome by
Agrobacterium
-mediated transformation. The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a substantial reduction in the levels of PG mRNA and enzymatic activity in ripening fruit. The steady-state levels of PG antisense RNA in green fruit of transgenic plants were lower than the levels of PG mRNA normally attained during ripening. However, analysis of transcription in isolated nuclei demonstrated that the antisense RNA construct was transcribed at a higher rate than the tomato PG gene(s). Analysis of fruit from transgenic plants demonstrated a reduction in PG mRNA and enzymatic activity of 70-90%. The reduction in PG activity did not prevent the accumulation of the red pigment lycopene.
cDNA encoding the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.
Authors: Hereld D , Hart G W , Englund P T
Published: May 2006

VSG lipase of Trypanosoma brucei specifically cleaves the glycosyl-phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein (VSG), releasing this protein from the plasma membrane. It also cleaves similar membrane anchors on some mammalian proteins. VSG lipase may play a role in processes such as parasite differentiation or antigenic variation. We describe here the cloning and sequencing of a cDNA encoding VSG lipase from T. brucei.
Structure and nucleotide sequence of the rat intestinal vitamin D-dependent calcium binding protein gene.
Authors: Krisinger J , Darwish H , Maeda N , DeLuca H F
Published: May 2006

The vitamin D-dependent intestinal calcium binding protein (ICaBP, 9 kDa) is under transcriptional regulation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonal active form of the vitamin. To study the mechanism of gene regulation by 1,25-(OH)2D3, we isolated the rat ICaBP gene by using a cDNA probe. Its nucleotide sequence revealed 3 exons separated by 2 introns within approximately 3 kilobases. The first exon represents only noncoding sequences, while the second and third encode the two calcium binding domains of the protein. The gene contains a 15-base-pair imperfect palindrome in the first intron that shows high homology to the estrogen-responsive element. This sequence may represent the vitamin D-responsive element involved in the regulation of the ICaBP gene. The second intron shows an 84-base-pair-long simple nucleotide repeat that implicates Z-DNA formation. Genomic Southern analysis shows that the rat gene is represented as a single copy.
The dominant non-gibberellin-responding dwarf mutant (
D8
) of maize accumulates native gibberellins
Authors: Fujioka Shozo , Yamane Hisakazu , Spray Clive R. , Katsumi Masayuki , Phinney Bernard O. , Gaskin Paul , MacMillan Jake , Takahashi Nobutaka
Published: May 2006

The endogenous gibberellins (GAs) were examined from young vegetative shoots of the dominant mutant,
Dwarf-8
, a GA-nonresponder, and normal maize; GA
44
, GA
17
, GA
19
, GA
20
, GA
29
, GA
1
, and GA
8
, members of the early-13-hydroxylation pathway, were identified from both kinds of shoots by full-scan mass spectra and Kovats retention indices. In addition, we report the identification of 3-epi-GA
1
, GA
3
, GA
4
, GA
5
, GA
7
, GA
9
, GA
12
, GA
15
, GA
24
, GA
34
, and GA
53
by using the same criteria. [1,7,12,18-
14
C
4
]GA
53
and -GA
44
, [17-
2
H
2
]GA
19
, and [17-
13
C,
3
H
2
]GA
20
, -GA
29
, -GA
1
, -GA
8
, and -GA
5
were used as internal standards to determine the endogenous levels of these GAs by measurement of isotope dilution, using capillary gas chromatography and selected ion monitoring. Shoots of
Dwarf-8
accumulate relatively high levels of GA
20
, GA
1
, and GA
8
. The accumulation of GA
1
appears to be related to gene dosage. Since
Dwarf-8
contains the same pattern of GAs as normals (including GA
1
and GA
3
), the genetic control point probably lies after GA
1
(and GA
3
). Thus
Dwarf-8
may be a GA receptor mutant or a mutant that controls a product downstream from the binding of the bioactive GA to a receptor.
Analysis of a T-cell tumor-specific breakpoint cluster at human chromosome 14q32.
Authors: Mengle-Gaw L , Albertson D G , Sherrington P D , Rabbitts T H
Published: May 2006

Cytogenetic abnormalities involving chromosome 14 band q32 are consistently observed in human T-cell tumors. Patients with ataxia-telangiectasia (AT) are especially prone to development of these tumors, which frequently carry either inversion inv(14)(q11;q32) or translocation t(14;14) (q11;q32) chromosomes. We have previously shown that the cytogenetic breakpoints of one t(14;14)(q11;q32) chromosome and two inv(14)(q11;q32) chromosomes in T-cell tumors from AT and non-AT patients join the T-cell receptor alpha chain locus, at chromosome band 14q11, with a region(s) at 14q32 centromeric of the immunoglobulin heavy chain variable region (VH) gene IGHV. We now show that these two inv(14) breakpoints are linked by 2.1 kb of germ-line 14q32 DNA and that the three breakpoints define, by in situ hybridization analysis, a single locus at chromosome band 14q32.1 located about 15-20 million base pairs on the centromeric side of the IGH locus. Sequence analysis of the 14q32.1 breakpoint regions indicates that abnormal recombination does not universally result from mistaken V-D-J joining (D = diversity region; J = joining region). Therefore, we invoke a tumor selection model to describe the role of the 14q32.1 locus in tumor development.
An analysis of T-cell receptor variable region gene expression in major histocompatibility complex disparate mice.
Authors: Bill J , Appel V B , Palmer E
Published: May 2006

To define the impact of major histocompatibility complex (MHC)-encoded glycoproteins on the selection of the T-cell receptor repertoire, we have determined the frequency with which T-cell receptor variable region (V alpha and V beta) genes are expressed in T cells from MHC disparate mice. Approximately 500 T-cell hybridomas were generated from each of three strains of MHC congenic mice [B10 (H-2b), B10.BR (H-2k), and B10.Q (H-2q)] by fusing mitogen-stimulated lymph node T cells with the thymoma BW5147. RNA was prepared from 1629 individual hybridomas and analyzed for the expression of 10 V alpha and 16 V beta gene families. These experiments reveal significant differences in the relative contributions of 1 V alpha gene family (V alpha 3) and several V beta gene segments (V beta 5.1, -5.2, -11, and -12) to the T-cell receptor repertoire of MHC disparate mice.
Expression of CD3-associated antigen-binding receptors on suppressor T cells.
Authors: Kuchroo V K , Steele J K , Billings P R , Selvaraj P , Dorf M E
Published: May 2006

Three suppressor T (Ts)-cell hybridomas specific for 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were selected for surface expression of cluster determinant 3 (CD3) by using antibody (anti-CD3) or antigen (NP-bovine serum albumin) panning procedures followed by cloning at limiting dilution. The CD3-selected Ts hybridomas showed a 1-2 logarithmic enrichment in suppressor activity when compared to the parent lines; they also specifically bound NP-coupled sheep red blood cells in rosette assays. This antigen-binding ability could be down-modulated by anti-CD3 antibody. Similarly, surface expression of CD3 was specifically down-modulated by preincubation of these hybridomas with antigen. Anti-CD3 monoclonal antibody under reducing conditions coprecipitated a broad band of 38-50 kDa associated with two CD3 (25 and 16 kDa) bands. T-cell receptor, anti-alpha-specific monoclonal antibody also immunoprecipitated a broad band in the 41 to 49-kDa region. The combined results suggest that, like helper and cytotoxic T lymphocytes, Ts cells also bear antigen-specific receptors associated with CD3 molecules.
Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor.
Authors: Sadaie M R , Rappaport J , Benter T , Josephs S F , Willis R , Wong-Staal F
Published: May 2006

Single nucleotide alterations were introduced into an infectious clone of human immunodeficiency virus type 1 to create a series of missense mutants in the tat coding region. Although mutations in a proline-rich region and a basic lysine-arginine-rich region resulted in wild-type phenotypes, five of six mutations in a cysteine-rich domain completely abolished tat activity and virus replication. One cysteine mutant retained tat activity but was negative for virus expression. Surprisingly, this mutant could not be complemented by tat, and virus expression was restored only by cotransfection with a plasmid expressing the rev gene. Another mutant with an alteration toward the C-terminal region showed significantly reduced tat activity and required complementation by a combination of tat and rev for virus replication. Further analysis revealed that a previously unrecognized splice acceptor site within this region, apparently used to generate the rev mRNA, had been altered. We provide evidence suggesting that tat and rev proteins are encoded by distinct mRNA species.
Effects of passive immunization in patients with the acquired immunodeficiency syndrome-related complex and acquired immunodeficiency syndrome.
Authors: Karpas A , Hill F , Youle M , Cullen V , Gray J , Byron N , Hayhoe F , Tenant-Flowers M , Howard L , Gilgen D
Published: May 2006

Infection with the human immunodeficiency virus type 1 (HIV-1) is usually followed by a vigorous immune response that temporarily protects against disease progression. After a variable asymptomatic period, acquired immunodeficiency syndrome (AIDS)-related complex (ARC) and AIDS develop in most infected individuals. We have demonstrated that healthy HIV-1-infected individuals have neutralizing antibodies and a high titer of antiviral antibodies. In contrast, AIDS patients have undetectable levels of neutralizing antibodies, low titers of antiviral antibodies, and, frequently, HIV p24 antigenemia. These observations prompted us to attempt passive immunization in ARC and AIDS patients. Ten consistently viral-antigen-positive patients (mean, greater than 6 months) were treated, resulting in sustained clearance of p24 antigen. Patients either maintained or increased their antiviral antibody titers. The raised titers result from increased antibody synthesis by the recipients. Circulating CD4+ cell counts were unchanged after 2 months. By the third month none of these patients remained in hospital. As this treatment was of minimal toxicity, it merits wider evaluation in ARC and AIDS patients.
Even transcriptionally competent proviruses are silent in bovine leukemia virus-induced sheep tumor cells.
Authors: Van den Broeke A , Cleuter Y , Chen G , Portetelle D , Mammerickx M , Zagury D , Fouchard M , Coulombel L , Kettmann R , Burny A
Published: May 2006

To investigate the role of proviral integration and expression in cellular transformation induced by bovine leukemia virus (BLV), three BLV-induced tumors harboring a single proviral copy were selected upon restriction and hybridization analysis. Tumors 344 and 395 were shown to contain a full-size proviral copy, whereas in tumor 1345 the provirus appeared to be heavily deleted. RNA gel blot hybridization with an antisense RNA probe showed no transcription of the viral sequences in the fresh tumors or in sheep tumor cells growing in vitro. The proviruses were cloned and transfected in mammalian cell lines. Transient-expression experiments revealed that the complete proviruses were still able to express the trans-activating protein (Tat) as well as structural proteins, demonstrating that the nonexpression of a provirus in a tumor cell does not necessarily imply a structural alteration of the viral information. In contrast, sequence analysis of the provirus with a large deletion and transient-expression assays proved that this truncated provirus, isolated from a tumor, was unable to code for viral proteins. These data indicate that expression of viral genes, including tat, is not required for the maintenance of the transformed state.
Fetal hemoglobin synthesis in vivo: direct evidence for control at the level of erythroid progenitors.
Authors: Umemura T , Al-Khatti A , Papayannopoulou T , Stamatoyannopoulos G
Published: May 2006

To test directly whether the control of fetal hemoglobin (HbF) in the adult takes place at the level of erythroid progenitors or at the level of erythroblasts, we treated animals with high doses of erythropoietin and examined the effects of this manipulation on the globin gene programs of erythroid progenitors. We found that administration of erythropoietin produced a rapid expansion of all classes of erythroid progenitors. Almost all the expansion of colony-forming units-erythroid and 46-56% of erythroid clusters was due to the increase of HbF-programmed erythroid progenitors. The expansion of HbF-programmed erythroid progenitors was followed, 2-3 days later, by a wave of reticulocytes containing HbF in the peripheral blood. These results provide direct in vivo evidence that fetal-globin expression in the adult is controlled at the level of erythroid progenitors.
Long-term potentiation differentially affects two components of synaptic responses in hippocampus.
Authors: Muller D , Lynch G
Published: May 2006

We have used low magnesium concentrations and the specific antagonist D-2-amino-5-phosphonopentanoate (D-AP5) to estimate the effects of long-term potentiation (LTP) on the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of postsynaptic responses. LTP induction resulted in a considerably larger potentiation of non-NMDA as opposed to NMDA receptor-related currents. Increasing the size of postsynaptic potentials with greater stimulation currents or with paired-pulse facilitation produced opposite effects; i.e., those aspects of the response dependent on NMDA receptors increased to a greater degree than did those components mediated by non-NMDA receptors. These results pose new constraints on hypotheses about the locus and nature of LTP and strongly suggest that postsynaptic modifications are part of the effect.
Charge configurations in viral proteins.
Authors: Karlin S , Brendel V
Published: May 2006

The spatial distribution of the charged residues of a protein is of interest with respect to potential electrostatic interactions. We have examined the proteins of a large number of representative eukaryotic and prokaryotic viruses for the occurrence of significant clusters, runs, and periodic patterns of charge. Clusters and runs of positive charge are prominent in many capsid and core proteins, whereas surface (glyco)proteins frequently contain a negative charge cluster. Significant charge configurations are abundant in regulatory proteins implicated in transcriptional transactivation and cellular transformation. Proteins with charge structures are much more predominant in animal DNA viruses as compared to animal RNA viruses and prokaryotic viruses. This contrast might reflect the role of protein charge structures in facilitating competitive virus-host interactions involving the cellular transcription, translation, protein sorting, and transport apparatus.
Osmotaxis in Escherichia coli.
Authors: Li C , Boileau A J , Kung C , Adler J
Published: May 2006

The escape of motile organisms from high concentrations of chemicals was studied in Escherichia coli. We have found all chemicals tested to be osmorepellents. It was shown in both a spatial assay and a temporal assay that the known sensory receptors for chemotaxis are not used for osmotaxis, so a different sensory mechanism appears to be employed. According to the temporal assay, the mechanism between sensory receptors and flagella is also not used for tumbling response (at least in solutions above 0.4 osmolar).
ATP sensitizes the insulin receptor to insulin.
Authors: Ridge K D , Hofmann K , Finn F M
Published: May 2006

Insulin receptor with high insulin binding and tyrosine kinase activities has been prepared from human placenta. Based on a molecular mass of 306 kDa for the receptor (the value obtained from the sum of the amino acid residues), this preparation is capable of binding 1.48 mol of insulin per mol of receptor. The receptor is free from phosphatase and ATPase activity and is not stimulated by sodium vanadate. Autophosphorylation is linear with respect to receptor concentration, and the 32P incorporated is stable even in the presence of a 100-fold excess of unlabeled ATP. The Km for ATP is 208 microM. N-Ethylmaleimide inhibits autophosphorylation. Alkylation with 3H-labeled N-ethylmaleimide results in the incorporation of 1.13 +/- 0.37 mol of N-ethylmaleimide per mol of insulin binding activity exclusively into the beta subunit of the receptor. The nonhydrolyzable ATP analog adenosine 5'-[beta,gamma-imido]triphosphate stimulates autophosphorylation of the receptor, an effect that is evident at ATP concentrations below 1 mM. The stimulatory effect of adenosine 5'-[beta,gamma-imido]triphosphate is the result of increasing the binding of insulin to the alpha subunit, and this reflects itself in a shift to the left of the insulin dose-response curve for autophosphorylation. The same is true for ATP. As a consequence, it is now possible to reconcile the concentration of insulin necessary for stimulating the autophosphorylation reaction with physiological levels and with the levels of insulin required for its classical biological effects.
Structural basis of beta-adrenergic receptor subtype specificity studied with chimeric beta 1/beta 2-adrenergic receptors.
Authors: Frielle T , Daniel K W , Caron M G , Lefkowitz R J
Published: May 2006

The beta 1- and beta 2-adrenergic receptors are two structurally related, but pharmacologically distinguishable, receptor subtypes, both of which activate adenylyl cyclase in a catecholamine-dependent manner through the guanine nucleotide-binding regulatory protein Gs. The receptors are approximately 50% identical in amino acid sequence and each is characterized by the presence of seven putative transmembrane domains. To elucidate the structural basis for the pharmacological distinctions between these two receptor subtypes, we constructed a series of chimeric beta 1/beta 2-adrenergic receptor genes and expressed them by injection of RNA into Xenopus laevis oocytes. The pharmacological properties of the expressed chimeric receptor proteins were assessed by radioligand binding and adenylyl cyclase assays utilizing subtype-selective agonists and antagonists. Our data indicate that transmembrane region IV is largely responsible for determining beta 1 vs. beta 2 properties with respect to agonist binding (relative affinities for epinephrine and norepinephrine). Transmembrane regions VI and VII play an important role in determining binding of beta 1 vs. beta 2 selective antagonists. However, a number of the other transmembrane regions also contribute, to a lesser extent, to the determination of beta-adrenergic receptor subtype specificity for agonists and antagonists. Thus, several of the membrane-spanning regions appear to be involved in the determination of receptor subtype specificity, presumably by formation of a ligand-binding pocket, with determinants for agonist and antagonist binding being distinguishable.
Catabolites of chlorophyll in senescing barley leaves are localized in the vacuoles of mesophyll cells
Authors: Matile Philippe , Ginsburg Stefan , Schellenberg Maja , Thomas Howard
Published: May 2006

Senescing barley leaves accumulate a series of pink pigments with the chemical properties of catabolites derived from chlorophyll. Levels of the major component of this group of pigments were quantified by HPLC and shown to be maximal in tissues exhibiting maximal rates of chlorophyll degradation. Protoplasts were isolated from senescent leaf tissue and fractionated to yield intact vacuoles and plastids. Although small but significant proportions both of total catabolites and of the dominant component of the series were recovered from the plastid fraction, the vast bulk of these compounds could be assigned to the vacuole. These observations suggest a role for the vacuole in the later stages of chlorophyll breakdown during senescence.
Lineage-specific regulation of the vasoactive intestinal peptide gene in neuroblastoma cells is conferred by 5.2 kilobases of 5'-flanking sequence.
Authors: Waschek J A , Hsu C M , Eiden L E
Published: May 2006

The expression of a transfected plasmid containing 5.2 kilobases (kb) of 5' regulatory DNA sequence of the human vasoactive intestinal peptide (VIP) gene attached to coding sequences of the reporter gene chloramphenicol acetyltransferase (CAT) was compared with endogenous VIP expression in subclones of the human neuroblastoma cell line SK-N-SH. These subclones vary widely in basal and inducible quantities of VIP and its precursor mRNA and can be interconverted under specified culture conditions. Endogenous VIP immunoreactivity, detectable in all subclones, was lowest in the neuronal subclone SH-SY-5Y, whereas 15- to 25-fold higher levels were observed in the epithelial-appearing SH-EP and intermediate SH-IN subclones. Treatment with 10 nM phorbol 12-myristate 13-acetate (PMA) stimulated VIP peptide levels approximately 5-fold in SH-SY-5Y cells but did not increase appreciably VIP levels in the other subclones. Treatment with 2.5 microM forskolin resulted in less than 50% stimulation of VIP expression in all subclones. Levels of mRNA encoding the VIP precursor generally paralleled these differences in VIP immunoreactivity. In cells transfected with the VIP/CAT fusion gene, CAT activity reflected closely these differences in basal VIP expression and the changes in response to PMA and forskolin. Deletion of 2.7 kb of the most upstream sequences resulted in an 80-90% reduction in basal CAT activity in SH-IN, but not SH-SY-5Y cells, and resulted in an 80% reduction in PMA stimulation in SH-SY-5Y cells. Deletion to within 74 nucleotides of the transcription start site resulted in CAT expression in SH-IN cells that was only 3% of that seen with the full 5.2-kb flanking sequences and further diminished the remaining PMA responsiveness in SH-SY-5Y cells. The data indicate that important cell-type-specific transcription regulatory sequences reside greater than 2.5 kb upstream from the VIP transcription start site.