bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a genomic hybrid specifying the human erythrocyte antigen Dantu: Dantu gene is duplicated and linked to a delta glycophorin gene deletion.
Authors: Huang C H , Blumenfeld O O
Published: May 2006

Dantu is a rare antigen of the human MNSs blood group system carried by a hybrid glycophorin on the erythrocyte membrane. To delineate its structure and relationship to alpha and delta glycophorins at the genomic level, we analyzed DNA from a three-generation family, in which both the presence of Dantu and Mi-III (another rare MNSs antigen) and the absence of delta glycophorin were seen. Three gross alterations in the gene cluster correlated with the observed phenotypes. Differential hybridization, secondary restriction analysis, and sequence-specific oligonucleotide probing established that Dantu glycophorin is encoded by a distinct hybrid gene derived from fusion of delta and alpha genes. The junction point of the Dantu gene was assigned to the region spanning codons 31-39 of delta and codons 72-79 of alpha genes. Dosage quantitation suggested that Dantu gene is duplicated and tandemly arranged. Identification of delta gene deletion disclosed the molecular basis for the absence of delta glycophorin in two Dantu and Mi-III double heterozygotes. The DNA inheritance pattern together with DNA typing of MN blood groups verified the genotypes and established that the duplicated Dantu hybrid gene is linked to alpha M gene as well as a delta gene deletion. The origin of such a haplotype in a segment of Black population would require two recombination events via either unequal homologous crossing-over or gene conversion.
Genome rearrangements activate the Epstein-Barr virus gene whose product disrupts latency.
Authors: Rooney C , Taylor N , Countryman J , Jenson H , Kolman J , Miller G
Published: May 2006

A defective Epstein-Barr virus (EBV) containing a deleted and rearranged genome (het DNA) causes latent EBV to replicate. This activity maps to the 2.7-kilobase-pair WZhet fragment. The BZLF1 open reading frame, present within WZhet as well as in the standard viral BamHI Z fragment, encodes the protein ZEBRA, which induces viral replication. Using gene transfers into Burkitt lymphoma cells, we now demonstrate that rearranged sequences juxtaposed to BZLF1 in het DNA facilitate expression of ZEBRA protein. Two stretches of EBV sequences within a palindromic region of het DNA contain positive regulatory elements. One set, derived from the viral large internal repeat, is newly positioned upstream of BZLF1; the second set is downstream of BZLF1 in het DNA. The capacity of defective HR-1 viruses to disrupt latency of the standard EBV genome is due to abnormal regulation of the BZLF1 gene as a result of genomic rearrangements.
Site-specific semisynthetic variant of human hemoglobin.
Authors: Hefta S A , Lyle S B , Busch M R , Harris D E , Matthew J B , Gurd F R
Published: May 2006

A single round of Edman degradation was employed to remove the NH2-terminal valine from isolated alpha chains of human hemoglobin. Reconstitution of normal beta chains with truncated or substituted alpha chains was used to form truncated (des-Val1-alpha 1) and substituted ([[1-13C]Gly1]alpha 1) tetrameric hemoglobin analogs. Structural homology of the analogs with untreated native hemoglobin was established by using several spectroscopic and physical methods. Functional studies indicate that the reconstituted tetrameric protein containing des-Val1-alpha chains has a higher affinity for oxygen, is less influenced by chloride ions or 2,3-bisphosphoglycerate, and shows lower cooperativity than native hemoglobin. These results confirm the key functional role of the alpha-chain NH2 terminus in mediating cooperative oxygen binding across the dimer interface. The NH2-terminal pK1/2 value was determined for the [13C]glycine-substituted analog to be 7.46 +/- 0.09 at 15 degrees C in the carbon monoxide-liganded form. This value, measured directly by 13C NMR, agrees with the determination made by the less-direct 13CO2 method and confirms the role of this residue as a contributor to the alkaline Bohr effect; however, it is inconsistent with the presence of an NH2-terminal salt bridge to the carboxylate of Arg-141 of the alpha chain in the liganded form.
Properties of a high-affinity DNA binding site for estrogen receptor.
Authors: Peale F V , Ludwig L B , Zain S , Hilf R , Bambara R A
Published: May 2006

It has been shown previously that a short sequence from the 5' regulatory region of the Xenopus laevis vitellogenin gene A2, when appropriately placed, can confer estrogen responsiveness to another gene. Using the Xenopus sequence and similar sequences from the 5' regulatory regions of other estrogen-responsive genes, we derived a consensus sequence 38 nucleotides long. The sequence contains an inverted repeat (5' C-A-G-G-T-C-A-G-A-G-T-G-A-C-C-T-G 3') and an A/T-rich region. Plasmids carrying a single copy of the sequence bound 3-fold-more partially purified estrogen receptor (ER) than did control plasmids when assayed by gel filtration. Maximum specificity for ER binding occurs at 100-150 mM ionic strength and pH 7.5-8.0. Plasmids carrying multiple copies of the sequence bound correspondingly more ER. The dissociation constant for ER bound to the sequence is 0.5 nM. This value is lower by a factor of about 400 than the dissociation constant for ER bound to an equivalent length of plasmid DNA. Portions of the consensus sequence were evaluated for binding efficiency. Plasmids containing the inverted repeat alone bound ER, though less efficiently than did plasmids containing the entire sequence. The A/T-rich region alone was ineffective in binding ER. Linearization of the plasmid DNA did not enhance specific binding efficiency for ER. This model system represents an effective tool for characterization of ER binding to DNA sequences involved in the regulation of gene expression.
Sequence of the human 40-kDa keratin reveals an unusual structure with very high sequence identity to the corresponding bovine keratin.
Authors: Eckert R L
Published: May 2006

The complete amino acid and DNA sequences of the human 40-kDa keratin are reported. The DNA sequence encodes a protein of 44,098 Da, which is unique in that it lacks the terminal non-alpha-helical tail segment found in all other keratins. When the human 40-kDa keratin amino acid sequence is compared to the corresponding bovine keratin, the overall identity is 89%. The coil-forming regions are 89% identical and the head regions are 88% identical. This similarity is also evident in the DNA sequence of the coding region, the 5' upstream sequences, and the 3' noncoding sequences. The high degree of cross-species identity between bovine and human 40-kDa keratins suggests that there is strong evolutionary pressure to conserve the structure of this keratin. This in turn suggests an important and universal role for this intermediate filament subunit in all species.
Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenografts in nude mice.
Authors: Yang H M , Reisfeld R A
Published: May 2006

Doxorubicin (DXR) was covalently conjugated to a monoclonal antibody (mAb), 9.2.27 (IgG2a), which recognizes a chondroitin sulfate proteoglycan expressed preferentially on the surface of human melanoma cells. Immunoconjugates with a molar ratio of DXR to mAb ranging from 2:1 to 10:1 were obtained by coupling the drug via an acid-sensitive linker, cis-aconitic anhydride. The immunoreactivity of mAb 9.2.27 was well retained after conjugation. DXR-mAb 9.2.27 conjugates were found to be 2 orders of magnitude more potent in killing tumor cells in vitro (IC50 = 0.1 microM) than free drug targeted to drug receptor(s). Most significantly, DXR-mAb 9.2.27 immunoconjugates specifically suppressed the growth of established tumors in vivo and prolonged the life-span of tumor-bearing nude mice. This suppression of melanoma growth achieved by the immunoconjugate was both tumor and antibody specific. A biodistribution study indicated that DXR-mAb 9.2.27 conjugates delivered at least 4 times more DXR (3.7% total injected dose per g of tumor) as compared to free DXR alone (0.8% total injected dose per g of tumor) in tumor-bearing nude mice 48 hr postinjection. The tumor-suppressive effects of DXR-mAb 9.2.27 conjugates are even more remarkable since free DXR did not suppress tumor growth in vivo and also because this drug per se is known to be quite ineffective for the treatment of human melanoma.
Isolation of a cDNA clone for the gamma subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: import and cleavage of the precursor protein.
Authors: Yu L M , Merchant S , Theg S M , Selman B R
Published: May 2006

A cDNA library from Chlamydomonas reinhardtii, constructed in the phage expression vector lambda gt11, was probed with antiserum directed against the nuclear-encoded gamma subunit of the chloroplast H+-transporting ATP synthase [ATP phosphohydrolase (H+-transporting) or chloroplast coupling factors 0 and 1, EC 3.6.1.34] of C. reinhardtii. A cDNA was isolated and transcribed in vitro. The transcript was translated in vitro and immunoprecipitated with anti-gamma-subunit serum to yield a product that coelectrophoresed with the immunoprecipitated product from in vitro-translated polyadenylylated RNA. These proteins were larger than the mature gamma subunit, either immunoprecipitated as chloroplast coupling factor 1 or as the individual subunit. Thus, the gamma subunit is synthesized as a precursor of greater molecular weight in C. reinhardtii. Furthermore, the precursor protein encoded by the cDNA is imported into pea chloroplasts and processed to a lower molecular weight polypeptide that coelectrophoreses with mature C. reinhardtii gamma subunit. The largest cDNA isolated is about the same length as the corresponding mRNA (approximately equal to 1900 bases long) and probably contains the entire coding region. Southern blot analyses revealed restriction fragment length polymorphisms and that the gamma subunit is probably encoded by an intron-containing single-copy gene.
Induction and properties of syngeneic murine anti-immunoglobulin D.
Authors: Haba S , Thorbecke G J , Nisonoff A
Published: May 2006

High titers of autoantibodies directed to isotypic determinants of IgD were produced by inoculation of syngeneic monoclonal IgD, conjugated covalently to keyhole limpet hemocyanin, into adult or neonatal inbred mice. Anti-idiotypic antibodies were induced at the same time. The average affinity of the mouse antibodies (Ka approximately equal to 10(7) M-1) is similar to that of rabbit anti-IgD and of syngeneic anti-IgE induced by the same procedure. Results indicate that B cells of the mice are not tolerant to serum IgD and that tolerance is maintained at the level of T cells. Direct interaction of the syngeneic anti-IgD with cell-surface IgD was minimal, and there was no convincing evidence that cell-surface IgD was down-regulated in the anti-IgD-producing mice. Further studies, preferably employing monoclonal anti-IgD, are required to determine whether epitopes on cell-surface IgD can be recognized by syngeneic anti-IgD. The ability to generate in vivo high titers of anti-IgD should facilitate the production of such monoclonal antibodies.
Desensitization of rat renal thick ascending limb cells to vasopressin.
Authors: Elalouf J M , Sari D C , Roinel N , de Rouffignac C
Published: May 2006

Previous studies from this laboratory have demonstrated that vasopressin stimulates K, Mg, Ca, Cl, and Na reabsorption by the thick ascending limb of Henle's loop (TALH) of the rat kidney. Micropuncture of superficial nephrons and clearance experiments were performed to determine whether desensitization of the TALH to vasopressin may be demonstrated in vivo and whether such desensitization is specific for the effects of vasopressin (i.e., homologous) or also alters the response to the other hormones acting on the same pool of adenylate cyclase in this nephron segment. Brattleboro rats, with hereditary hypothalamic diabetes insipidus (DI), were given i.m. injections of 1-desamino-8-D-arginine-vasopressin (des-1-amino-[DArg8]VP (herein designated dDAVP); 2 micrograms/day) for 3 days. The effects of maximal physiological doses of arginine-8-vasopressin ([Arg8]VP (herein designated AVP); 20 pg/min per 100 g of body weight) were studied 2 days after the cessation of treatment, when the animals had returned to DI. The K, Mg, Ca, and, to a lesser extent, Cl and Na concentrations in the fluid leaving the TALH of superficial nephrons were higher in dDAVP-treated than in untreated rats given similar amounts of AVP during the experiments. A 50-60% desensitization of the TALH to AVP was still apparent 2 days after stopping the dDAVP injections. Desensitization is homologous, as judged from normal responses to physiological doses of glucagon and calcitonin, two hormones acting on the same cyclase pool as AVP in the rat TALH. The AVP-dependent increase of urine osmolality, however, indicated that its effects on the permeability to water of the collecting duct were scarcely affected in dDAVP-treated rats. It is concluded that (i) AVP induces homologous desensitization in the rat TALH and (ii) the TALH can be markedly desensitized to AVP when the collecting duct response to this hormone is poorly affected or even fully maintained.
Structure of the orthorhombic form of Mn
2
Al
7
, Fe
2
Al
7
, and (Mn
0.7
Fe
0.3
)
2
Al
7
that by twinning produces grains with decagonal point-group symmetry
Authors: Pauling Linus
Published: May 2006

Analysis of electron diffraction photographs of grains of Mn
2
Al
7
, Fe
2
Al
7
, and (Mn
0.7
Fe
0.3
)
2
Al
7
leads to the conclusion that they are 5-fold twins of a 1664-atom orthorhombic crystal with
a
= 32.86 Å,
b
= 31.23 Å, and
c
= 24.80 Å and with 16 icosahedral clusters of 104 atoms in positions shifted by small amounts from those of the cubic β-tungsten structure.
Directional mutation pressure and neutral molecular evolution.
Authors: Sueoka N
Published: May 2006

A quantitative theory of directional mutation pressure proposed in 1962 explained the wide variation of DNA base composition observed among different bacteria and its small heterogeneity within individual bacterial species. The theory was based on the assumption that the effect of mutation on a genome is not random but has a directionality toward higher or lower guanine-plus-cytosine content of DNA, and this pressure generates directional changes more in neutral parts of the genome than in functionally significant parts. Now that DNA sequence data are available, the theory allows the estimation of the extent of neutrality of directional mutation pressure against selection. Newly defined parameters were used in the analysis, and two apparently universal constants were discovered. Analysis of DNA sequence has revealed that practically all organisms are subject to directional mutation pressure. The theory also offers plausible explanations for the large heterogeneity in guanine-plus-cytosine content among different parts of the vertebrate genome.
High recombination between two physically close human basement membrane collagen genes at the distal end of chromosome 13q.
Authors: Bowcock A M , Hebert J M , Wijsman E , Gadi I , Cavalli-Sforza L L , Boyd C D
Published: May 2006

Two basement membrane collagen genes coding for the pro alpha 1 chain and pro alpha 2 chain of type IV collagen map to 13q34 and are linked with a maximum likelihood estimate of recombination of 0.028 at a logarithm of odds (lod) score of 19.98. The single-copy sequence that identifies the locus D13S3 is also closely linked to both collagen genes. Four enzymes reveal polymorphisms with COL4A1, and 10 haplotypes have been observed in Caucasoids. Within COL4A1 a nonrandom association of alleles exists only between alleles defined by Hae III and those defined by the other three enzymes. A random association of alleles of COL4A1 and COL4A2 is observed. Between the two collagen genes were detected three meiotic recombination events that contributed to the estimate of 2.8% recombination. This is higher than expected for two genes that lie within 650 kilobases of each other. The lack of linkage disequilibrium between COL4A1 and COL4A2 is in agreement with the relatively high recombination that is observed.
Determination of the absolute configuration of (-)-(2R)-succinic-2-d acid by neutron diffraction study: unambiguous proof of the absolute stereochemistry of the NAD+/NADH interconversion.
Authors: Yuan H S , Stevens R C , Fujita S , Watkins M I , Koetzle T F , Bau R
Published: May 2006

The absolute configuration of the CHD group (D = deuterium) in (-)-(2R)-succinic-2-d acid, as prepared from (-)-(2S,3R)-malic-3-d acid, has been shown unambiguously to be R by the technique of single-crystal neutron diffraction. The optically active cation (+)-phenylethylammonium was used as the chiral reference. The structure of [C6H5CH3CHNH3]+[HOOCCH2CHDCOO]- has been studied with x-ray diffraction at room temperature and neutron diffraction at 100 K. Crystal data from the neutron diffraction analysis of the phenylethylammonium salt of the title compound at 100 K: space group P21; a = 8.407(2) A, b = 8.300(4) A, c = 8.614(2) A, beta = 91.20(3) degrees; unit cell volume = 600.9(3) A3, zeta = 2 (numbers in parentheses are the estimated standard deviations). Final agreement factors are R(F2) = 0.0355 and R(wF2) = 0.0457 for 1690 independent neutron reflections and 297 parameters varied. The result confirms the stereochemistry of the malate/succinate transformation, as well as the NAD+/NADH interconversion, and demonstrates the usefulness of the single-crystal neutron diffraction method for determining the absolute configuration of molecules having a chiral monodeuteriomethylene group.
An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes.
Authors: Takahashi H , Cohen J , Hosmalin A , Cease K B , Houghten R , Cornette J L , DeLisi C , Moss B , Germain R N , Berzofsky J A
Published: May 2006

Because cytotoxic T lymphocytes (CTL) may be important for preventing direct cell-to-cell transmission of human immunodeficiency virus (HIV), the agent responsible for acquired immunodeficiency syndrome, we have begun to investigate the epitope specificity and immune response (Ir) gene control of anti-HIV CTL responses in experimental animals. Mice were infected with a recombinant vaccinia virus expressing the HIV gp160 envelope gene, and the primed lymphocytes were restimulated in vitro with a transfected histocompatible cell line expressing the same gene. Our results show that H-2d mice are CTL high responders and H-2k mice are low responders to the HIV gp160 envelope protein under these conditions. Moreover, the H-2d mice respond predominantly to a single immunodominant site represented by a 15-residue synthetic peptide conforming to the amphipathic alpha-helix model of T-cell epitopes and seen by CD4- CD8+ CTL in association with the Dd class I major histocompatibility complex (MHC) molecules. The facts that CTL responses were detected in the context of only one of four class I MHC molecules tested and that the response was limited predominantly to a single epitope indicate that the CTL repertoire elicited by the HIV envelope protein in association with murine class I MHC molecules may be very limited. In addition, this epitope occurs in a highly variable segment of the envelope protein. This puts constraints on the use of a single peptide sequence from this region in a vaccine, as such a vaccine would have to be polyvalent. Nevertheless, this same variability suggests that this region may be under selective pressure from human CTL, and therefore that this site may be immunodominant in humans as well as mice and so of clinical importance in vaccine development.
Evidence for selenocysteine coordination to the active site nickel in the [NiFeSe]hydrogenases from Desulfovibrio baculatus.
Authors: Eidsness M K , Scott R A , Prickril B C , DerVartanian D V , Legall J , Moura I , Moura J J , Peck H D
Published: May 2006

Ni and Se x-ray absorption spectroscopic studies of the [NiFeSe]hydrogenases from Desulfovibrio baculatus are described. The Ni site geometry is pseudo-octahedral with a coordinating ligand composition of 3-4 (N,O) at 2.06 A, 1-2 (S,Cl) at 2.17 A, and 1 Se at 2.44 A. The Se coordination environment consists of 1 C at 2.0 A and a heavy scatterer M (M = Ni or Fe) at approximately 2.4 A. These results are interpreted in terms of a selenocysteine residue coordinated to the Ni site. The possible role of the Ni-Se site in the catalytic activation of H2 is discussed.
At least two mutant alleles of ornithine delta-aminotransferase cause gyrate atrophy of the choroid and retina in Finns.
Authors: Mitchell G A , Brody L C , Sipila I , Looney J E , Wong C , Engelhardt J F , Patel A S , Steel G , Obie C , Kaiser-Kupfer M
Published: May 2006

Gyrate atrophy of the choroid and retina (GA) is an inherited chorioretinal degeneration caused by deficiency of ornithine delta-aminotransferase (OAT; L-ornithine: 2-oxo-acid aminotransferase; EC 2.6.1.13). GA is one of the "Finnish genetic diseases," a group of several rare monogenic disorders that occur with increased frequency in the Finnish population. Using a combination of RNase A protection, genomic cloning, and polymerase chain reaction amplification of genomic DNA, we found one of two missense mutant OAT alleles to be present in each of 16 Finnish GA pedigrees. The first mutation R180T, in which arginine-180 is replaced by threonine, was present in homozygous form in patients from two pedigrees. The second mutation L402P, in which leucine-402 is replaced by proline, was present in homozygous form in patients from 14 pedigrees. Neither mutation was present in 19 Finnish controls. L402P was not present in 18 non-Finnish GA patients but R180T was found in an American GA patient. We constructed full-length mutant cDNAs by amplifying patient cDNA with the polymerase chain reaction and cloning a restriction fragment containing the mutation into an otherwise normal human OAT cDNA. These mutant cDNAs were then expressed in CHO-K1 cells, which lack endogenous OAT. Both R180T and L402P inactivate OAT. These results show molecular heterogeneity in GA alleles even in the Finnish population.
Class I major histocompatibility complex-restricted cytolytic T lymphocytes recognize a limited number of sites on the influenza hemagglutinin.
Authors: Braciale T J , Sweetser M T , Morrison L A , Kittlesen D J , Braciale V L
Published: May 2006

Two distinct regions of the influenza A/JAP/305/57 hemagglutinin molecule are identifiable as sites recognized by murine class I major histocompatibility complex (MHC) (H-2d)-restricted cytolytic T lymphocytes (CTL) generated in response to immunization with infectious type A influenza virus. Each of these sites can be mimicked by a synthetic oligopeptide of approximately 20 amino acids. Data presented herein indicate that these two sites define the dominant immunogenic epitopes on the hemagglutinin recognized by H-2Kd-restricted CTL. These same sites are not efficiently recognized by hemagglutinin-specific class I MHC-restricted CTL of several unrelated MHC haplotypes. These observations show that even for a large complex glycoprotein molecule like the influenza hemagglutinin, only a limited number of class I CTL recognition sites are generated in the infected cell and that the subset of immunogenic epitopes is dependent on the MHC haplotype of the responding individual. These parameters need to be considered in the design of synthetic and recombinant vaccines.
Overexpression of amyloid precursor protein A4 (beta-amyloid) immunoreactivity in genetically transformed cells: implications for a cellular model of Alzheimer amyloidosis.
Authors: Marotta C A , Chou W G , Majocha R E , Watkins R , LaBonne C , Zain S B
Published: May 2006

Among the major obstacles to clarifying molecular mechanisms involved in amyloid metabolism in Alzheimer disease has been the unavailability of laboratory models for this uniquely human disorder. The present studies were aimed at establishing genetically engineered cell lines that overexpress amyloid immunoreactivity and that may be relevant to amyloid accumulation in the Alzheimer disease brain. We used cloned amyloid cDNA that contains a region encoding A4 (beta-polypeptide) amino acids along with recently developed tumor virus vectors derived from simian virus 40 to prepare transformed cells. After transient and permanent transfection, a variety of cell types overexpressed A4 immunoreactivity that was detected by highly specific monoclonal antibodies. We observed that the use of an amyloid subdomain containing the A4 region, but lacking the sequence of a Kunitz-type protease inhibitor found in amyloid precursor protein variants, was sufficient to obtain cells that overproduced an A4 epitope. The transformed cells were readily propagated in culture and may provide an experimental medium to elucidate aspects of the molecular pathogenesis of Alzheimer disease. The cellular models may also serve as tools for deriving potentially useful therapeutic agents.
Tn7 transposition: recognition of the attTn7 target sequence.
Authors: Waddell C S , Craig N L
Published: May 2006

The bacterial transposon Tn7 encodes two distinct but overlapping transposition pathways. tnsABC + tnsD promote transposition to a specific site, attTn7, while tnsABC + tnsE promote transposition to many other sites unrelated to attTn7. We have identified a tnsD-dependent DNA binding activity that specifically recognizes attTn7. We have localized the recognition sequences for this activity to a 28-base-pair region and have shown that this same region can provide specific properties of an attTn7 target in vivo. Interestingly, these sequences are positioned more than 25 base pairs from the specific point of Tn7 insertion.
Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372----histidine).
Authors: Arai M , Inaba H , Higuchi M , Antonarakis S E , Kazazian H H , Fujimaki M , Hoyer L W
Published: May 2006

An immunoadsorbent method has been developed for the direct analysis of normal and variant plasma factor VIII. Using this method, the molecular defect responsible for mild hemophilia A has been identified for a patient whose plasma factor VIII activity is 0.05 unit/ml, even though the factor VIII antigen content is 3.25 units/ml. Although the variant factor VIII has an apparently normal molecular mass and chain composition, the 92-kDa heavy chain accumulates when the variant protein is incubated with thrombin and the 44-kDa heavy chain fragment cannot be detected. In contrast, thrombin cleavage of the 80-kDa light chain to the 72-kDa fragment is normal. As these data indicate a loss of factor VIII cleavage by thrombin at arginine-372, the genetic defect was determined by polymerase-chain-reaction amplification of exon 8 of the factor VIII gene and direct sequencing of the amplified product. A single-base substitution (guanine----adenine) was identified that produces an arginine to histidine substitution at amino acid residue 372. These data identify the molecular basis of an abnormal factor VIII, "factor VIII-Kumamoto," that lacks procoagulant function because of impaired thrombin activation.
Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain.
Authors: Simmer J P , Kelly R E , Scully J L , Grayson D R , Rinker A G , Bergh S T , Evans D R
Published: May 2006

Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.
Regulation of transcription by cyclic AMP-dependent protein kinase.
Authors: Mellon P L , Clegg C H , Correll L A , McKnight G S
Published: May 2006

cAMP-dependent protein kinase (PKA; ATP: protein phosphotransferase; EC 2.7.1.37) appears to be the major mediator of cAMP responses in mammalian cells. We have investigated the role of PKA subunits in the regulation of specific genes in response to cAMP by cotransfection of wild-type or mutant subunits of PKA together with cAMP-inducible reporter genes. Overexpression of catalytic subunit induced expression from three cAMP-regulated promoters (alpha-subunit, c-fos, E1A) in the absence of elevated levels of cAMP but did not affect expression from two unregulated promoters (Rous sarcoma virus, simian virus 40). Cotransfection of a regulatory subunit gene containing mutations in both cAMP binding sites strongly repressed both basal and induced expression from the cAMP-responsive alpha-subunit promoter without affecting expression from the Rous sarcoma virus promoter. These experiments indicate that cAMP induces gene expression through phosphorylation by the catalytic subunit and that the ambient degree of phosphorylation dictates the level of basal as well as induced expression of the cAMP-regulated alpha-subunit gene.
Modern mammal origins: evolutionary grades in the Early Cretaceous of North America.
Authors: Jacobs L L , Winkler D A , Murry P A
Published: May 2006

Major groups of modern mammals have their origins in the Mesozoic Era, yet the mammalian fossil record is generally poor for that time interval. Fundamental morphological changes that led to modern mammals are often represented by small samples of isolated teeth. Fortunately, functional wear facets on teeth allow prediction of the morphology of occluding teeth that may be unrepresented by fossils. A major step in mammalian evolution occurred in the Early Cretaceous with the evolution of tribosphenic molars, which characterize marsupials and placentals, the two most abundant and diverse extant groups of mammals. A tooth from the Early Cretaceous (110 million years before present) of Texas tests previous predictions (based on lower molars) of the morphology of upper molars in early tribosphenic dentitions. The lingual cusp (protocone) is primitively without shear facets, as expected, but the cheek side of the tooth is derived (advanced) in having distinctive cusps along the margin. The tooth, although distressingly inadequate to define many features of the organism, demonstrates unexpected morphological diversity at a strategic stage of mammalian evolution and falsifies previous claims of the earliest occurrence of true marsupials.
A human immunoglobulin G receptor exists in both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms.
Authors: Scallon B J , Scigliano E , Freedman V H , Miedel M C , Pan Y C , Unkeless J C , Kochan J P
Published: May 2006

Several cDNA clones encoding the human immunoglobulin G receptor CD16 were isolated from human lung or peripheral blood leukocyte cDNA libraries. Nucleotide sequence comparisons revealed that the cDNAs could be divided into two groups. cDNA clones in one group encode a protein that terminates 4 amino acids after the putative transmembrane domain. Clones in the second group encode a protein with an extra 21 amino acids that could comprise a cytoplasmic domain. Direct peptide sequencing was used to determine the N terminus of the mature CD16 receptor protein and supported the existence of the two forms of the receptor. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C resulted in the release of a large percentage of the CD16 molecules from the cell surface. In contrast, treatment of natural killer cells with phosphatidylinositol-specific phospholipase C did not release any CD16 from the cell surface. These data demonstrate that both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms of the CD16 molecule exist and that they are differentially expressed on neutrophils and natural killer cells.
Hydrins, hydroosmotic neurohypophysial peptides: osmoregulatory adaptation in amphibians through vasotocin precursor processing.
Authors: Rouillé Y , Michel G , Chauvet M T , Chauvet J , Acher R
Published: May 2006

From neurointermediate pituitary glands of Xenopus laevis and Rana esculenta, previously unreported peptides termed hydrins, active on water permeability of frog urinary bladder and frog skin (Brunn or "water-balance" effect), have been isolated and sequenced. These peptides seem to be derived from the pro-vasotocin-neurophysin precursor. Hydrin 1, found in Xenopus, has been identified as vasotocin C-terminally extended with the Gly-Lys-Arg sequence; hydrin 2, found in Rana, has been identified as vasotocin C-terminally extended with glycine. Hydrin 2 has been detected in several Ranidae (R. esculenta, Rana temporaria, Rana pipiens) and Bufonidae (Bufo bufo, Bufo ictericus) and appears to have a large distribution in terrestrial or semiaquatic anurans. Hydrins, in contrast to vasotocin, are not active on rat uterus or rat blood pressure. They are absent from other vasotocin-bearers such as birds and could be involved specifically in water-electrolyte regulation of amphibians.
Restoration of myelin formation by a single type of myelin basic protein in transgenic shiverer mice.
Authors: Kimura M , Sato M , Akatsuka A , Nozawa-Kimura S , Takahashi R , Yokoyama M , Nomura T , Katsuki M
Published: May 2006

A minigene containing mouse cDNA coding for the smallest type of myelin basic protein and including the native promoter was constructed and used to produce transgenic shiverer mice. The hypomyelinating mouse, the shiverer, has a deletion in its myelin basic protein gene, lacks all four types of myelin basic protein in its myelin, and shows abnormal behavior such as violent tremors. Five of twenty-one transgenic shiverer mice showed recovered protein synthesis, compact myelin formation, and normal behavior. These results suggest that a single type of myelin basic protein restores myelin formation and returns the shivering phenotype to normal in the transgenic shiverer mouse.
Self-assembly of synthetic phytochrome holoprotein
in vitro
Authors: Lagarias J. Clark , Lagarias Donna M.
Published: May 2006

The phytochrome holoprotein of plants requires a covalently bound linear tetrapyrrole (bilin) prosthetic group for its photoreceptor function. Here we show that synthetic phytochrome apoprotein prepared by transcription and translation of an
Avena
phytochrome cDNA construct combines
in vitro
with phycocyanobilin, an analog of the natural chromophore, to produce a photoactive holoprotein. These results indicate that holoprotein assembly is an “autocatalytic” process.
Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited.
Authors: Sarnow P
Published: May 2006

All cellular cytoplasmic mRNAs carry a 7-methylguanylate cap attached to their 5' ends. This cap structure is recognized by cap-binding proteins that then direct the binding of ribosomal subunits to this 5'-end complex. Poliovirus, a plus-stranded RNA virus, interferes with this cellular translation process by proteolytically inactivating the cap-binding protein complex. Subsequently the viral mRNA can be translated by an initiation process in which ribosomes bind internally to the mRNA [Pelletier, J. & Sonenberg, N. (1988) Nature (London) 334, 320-325], obviating cap-dependent translation. At least one cellular mRNA, encoding a heat shock-like protein, glucose-regulated protein 78/immunoglobulin heavy-chain binding protein, has been discovered to be translated at an increased rate in poliovirus-infected cells at a time when the translation of other cellular mRNAs is inhibited. The glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA thus exemplifies a cellular mRNA that is translated at a specifically enhanced rate by an as-yet-unresolved cap-independent initiation process in cells when the cap-binding protein complex is not functional.
Enzymes work by solvation substitution rather than by desolvation.
Authors: Warshel A , Aqvist J , Creighton S
Published: May 2006

Considerable attention has recently been drawn to the hypothesis that enzymes catalyze their reactions by displacing solvent and creating an environment similar to the gas phase for the reacting substrates. This "desolvation hypothesis" is reexamined in this paper by defining a common reference energy for reactions in various environments. It is argued that consistent attempts to describe the actual energetics of enzymatic reactions, taking either gas phase or solution as a reference, would contradict the above hypothesis. That is, the enzyme does remove water molecules from its substrate, but substitutes these molecules for another polar environment (namely, its active site). By taking amide hydrolysis as an example, we use experimentally estimated solvation energies and analyze the reaction profile in the gas phase, in solution, and in enzyme active sites. We show that the gas-phase reaction is characterized by an enormous activation barrier (associated with forming the charged nucleophile from neutral fragments), although the nucleophilic attack is essentially barrierless. On the other hand, the enzyme and solution reactions are found to have similar reaction profiles, with a lower activation barrier for the enzymatic reaction. Presumably, the fact that previous analyses of this problem did not involve the construction of the relevant thermodynamic cycles (and quantitative estimates of the corresponding solvation energies) led to the desolvation hypothesis. Our conclusion is that enzyme active sites provide specific polar environments that do not resemble the gas phase but that are designed for electrostatic stabilization of ionic transition states and that "solvate" these states more than water does.
Chromosome mapping of the human gene encoding the 68-kDa nuclear antigen (p68) by using the polymerase chain reaction.
Authors: Iggo R , Gough A , Xu W , Lane D P , Spurr N K
Published: May 2006

Chromosome mapping by Southern analysis of DNA from somatic cell hybrids is often unsuccessful when only cDNA probes are available. p68 is a putative RNA helicase that is antigenically related to the simian virus 40 large tumor antigen. By using the polymerase chain reaction and oligode-oxynucleotide primers based on the cDNA sequence, we have identified introns in the p68 gene. Comparison of human and mouse DNA fragments amplified with these primers revealed length differences that allowed us to identify the human gene. Application of this technique to DNA from human-mouse somatic cell hybrids and cell lines derived from them by chromosome-mediated gene transfer allowed us to map p68 to the distal part of the long arm of chromosome 17.
The beta-amyloid protein precursor of Alzheimer disease has soluble derivatives found in human brain and cerebrospinal fluid.
Authors: Palmert M R , Podlisny M B , Witker D S , Oltersdorf T , Younkin L H , Selkoe D J , Younkin S G
Published: May 2006

In this study, we use antisera to synthetic beta-amyloid protein precursor (beta APP) peptides to identify, in human brain and cerebrospinal fluid (CSF), soluble approximately 125- and approximately 105-kDa derivatives of the beta APP that lack the carboxyl terminus of the full-length, membrane-associated forms. We show that the soluble approximately 125-kDa beta APP derivative contains the Kunitz protease inhibitor domain, whereas the approximately 105-kDa form does not, and we confirm that these two proteins are soluble beta APP derivatives by purifying each from human CSF and directly sequencing its amino terminus.
U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA.
Authors: Maser R L , Calvet J P
Published: May 2006

U3 small nuclear RNA is hydrogen-bonded to high molecular weight nucleolar RNA and can be isolated from greater than 60S pre-ribosomal ribonucleoprotein particles, suggesting that it is involved in processing of ribosomal RNA precursors (pre-rRNA) or in ribosome biogenesis. Here we have used in vivo psoralen cross-linking to identify the region of pre-rRNA interacting with U3 RNA. Quantitative hybridization selection/depletion experiments with clones of rRNA-encoding DNA (rDNA) and cross-linked nuclear RNA showed that all of the cross-linked U3 RNA was associated with a region that includes the external transcribed spacer (ETS) at the 5' end of the human rRNA precursor. To further identify the site of interaction within the approximately 3.7-kilobase ETS, Southern blots of rDNA clones were sandwich-hybridized with cross-linked RNA and then probed for cross-linked U3 RNA. These experiments showed that U3 RNA was cross-linked to a 258-base sequence between nucleotides +438 and +695, just downstream of the ETS early cleavage site (+414). The localization of U3 to this region of the rRNA precursor was not expected from previous models for a base-paired U3-rRNA interaction and suggests that U3 plays a role in the initial pre-rRNA processing event.
5' flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Authors: Darville M I , Crepin K M , Hue L , Rousseau G G
Published: May 2006

The synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis, are catalyzed by 6-phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46), respectively. In liver, these two activities belong to separate domains of the same 470-residue polypeptide. Various mRNAs have been described for this bifunctional enzyme, which is controlled by hormonal and metabolic signals. To understand the origin and regulation of these mRNAs, we have characterized rat genomic clones encoding the liver isozyme, which is regulated by cAMP-dependent protein kinase, and the muscle isozyme, which is not. We describe here a 55-kilobase gene that encodes these isozymes by alternative splicing from two promoters. Each of the putative promoters was sequenced over about 3 kilobases and found to include nucleotide motifs for binding regulatory factors. The two isozymes share the same 13 exons and differ only by the first exon that, in the liver but not in the muscle isozyme, contains the serine phosphorylated by cAMP-dependent protein kinase. The gene was assigned to the X chromosome. An analysis of the exon limits of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in relation to its functional domains and to its similarity with other proteins plus its G + C content at the third codon position suggests that this gene originates from several fusion events.
Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.
Authors: Bordignon C , Yu S F , Smith C A , Hantzopoulos P , Ungers G E , Keever C A , O'Reilly R J , Gilboa E
Published: May 2006

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.
Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli.
Authors: Tauler A , Lange A J , el-Maghrabi M R , Pilkis S J
Published: May 2006

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.