bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide and predicted amino acid sequences of cloned human and mouse preprocathepsin B cDNAs.
Authors: Chan S J , San Segundo B , McCormick M B , Steiner D F
Published: May 2006

Cathepsin B is a lysosomal thiol proteinase that may have additional extralysosomal functions. To further our investigations on the structure, mode of biosynthesis, and intracellular sorting of this enzyme, we have determined the complete coding sequences for human and mouse preprocathepsin B by using cDNA clones isolated from human hepatoma and kidney phage libraries. The nucleotide sequences predict that the primary structure of preprocathepsin B contains 339 amino acids organized as follows: a 17-residue NH2-terminal prepeptide sequence followed by a 62-residue propeptide region, 254 residues in mature (single chain) cathepsin B, and a 6-residue extension at the COOH terminus. A comparison of procathepsin B sequences from three species (human, mouse, and rat) reveals that the homology between the propeptides is relatively conserved with a minimum of 68% sequence identity. In particular, two conserved sequences in the propeptide that may be functionally significant include a potential glycosylation site and the presence of a single cysteine at position 59. Comparative analysis of the three sequences also suggests that processing of procathepsin B is a multistep process, during which enzymatically active intermediate forms may be generated. The availability of the cDNA clones will facilitate the identification of possible active or inactive intermediate processive forms as well as studies on the transcriptional regulation of the cathepsin B gene.
Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells as revealed by monoclonal antibodies.
Authors: Hamada H , Tsuruo T
Published: May 2006

An overexpression of the plasma membrane glycoprotein of relative molecular size 170-180 kDa is consistently found in different multidrug-resistant human and animal cell lines, although the functional role of the protein in multidrug resistance is not known. Two monoclonal antibodies that interfere with biochemical functions were generated against the human myelogenous leukemia K-562 cells resistant to adriamycin (K-562/ADM). These antibodies, designated MRK16 and MRK17, are specifically reactive to K-562/ADM and a human ovarian cancer cell line resistant to adriamycin (2780AD). MRK16 modulated vincristine and actinomycin D transport in the resistant cells, while MRK17 specifically inhibited the growth of the resistant cells. Both antibodies recognized the 170- to 180-kDa glycoprotein. These data indicate that the 170- to 180-kDa glycoprotein is involved, directly or indirectly, in the drug transport mechanisms and the proliferation of multidrug-resistant tumor cell lines.
Differential expression of six light-harvesting chlorophyll a/b binding protein genes in maize leaf cell types.
Authors: Sheen J Y , Bogorad L
Published: May 2006

Bundle sheath chloroplasts of maize leaves contain about one-fourth as much light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP-II) as do mesophyll chloroplasts. We have determined that this difference is, in part, the result of differential expression of different LHCP-II genes. We have prepared and partially characterized cDNA clones specific for six LHCP-II genes of maize. Transcripts of these six LHCP-II genes are present at vastly different levels and account for about 95% of total LHCP-II mRNAs in bundle sheath and mesophyll cells of illuminated dark-grown maize leaves. Three genes are preferentially expressed in mesophyll cells, and their mRNAs constitute about 54% of the total LHCP-II transcripts in greening (24 hr) maize leaves. Two genes are expressed equally in bundle sheath and mesophyll cells. Most interestingly, the RNA of one gene that contributes about 8% of the total LHCP-II transcripts in leaves greening for 24 hr is present at a much higher level in bundle sheath than in mesophyll cells. Moreover, immunoblot analysis of maize thylakoids reveals at least five sizes of LHCP-II; these also differ from one another in their relative abundance in bundle sheath and mesophyll cells of developing maize leaves.
Random-clone strategy for genomic restriction mapping in yeast.
Authors: Olson M V , Dutchik J E , Graham M Y , Brodeur G M , Helms C , Frank M , MacCollin M , Scheinman R , Frank T
Published: May 2006

An approach to global restriction mapping is described that is applicable to any complex source DNA. By analyzing a single restriction digest for each member of a redundant set of lambda clones, a data base is constructed that contains fragment-size lists for all the clones. The clones are then grouped into subsets, each member of which is related to at least one other member by a significant overlap. Finally, a tree-searching algorithm seeks restriction maps that are consistent with the fragment-size lists for all the clones in each subset. The feasibility of the approach has been demonstrated by collecting data on 5000 lambda clones containing random 15-kilobase inserts of yeast DNA. It is shown that these data can be analyzed to produce regional maps of the yeast genome, extending in some cases for over 100 kilobases. In combination with hybridization probes to previously cloned genes, these local maps are already useful for defining the physical arrangement of closely linked genes. They may in the future serve as building blocks for the construction of a continuous global map.
Monoclonal and polyclonal antibodies against acetaldehyde-containing epitopes in acetaldehyde-protein adducts.
Authors: Israel Y , Hurwitz E , Niemelä O , Arnon R
Published: May 2006

Immunization of mice with acetaldehyde conjugated to human plasma proteins resulted in the production of polyclonal antibodies that reacted with erythrocyte protein-acetaldehyde conjugates, but not with control erythrocyte proteins. Such antibodies recognized erythrocyte protein-acetaldehyde conjugates prepared with 20-100 microM acetaldehyde, concentrations that exist in the blood of alcoholics. The antibodies also recognized acetaldehyde condensation products with synthetic poly-(L-lysine). Immunization with keyhole limpet hemocyanin-acetaldehyde conjugates resulted in antibodies against both plasma protein-acetaldehyde and erythrocyte protein-acetaldehyde conjugates, which did not cross-react with the respective unmodified carrier proteins. Immunization with human erythrocyte protein-acetaldehyde condensates led to the production of antibodies against both the protein moiety as well as the condensate. Monoclonal antibodies with affinities 50 times greater for the condensate than for the carrier protein were produced by hybridization of spleen cells from the immunized mice. Chronic alcohol administration to mice for 45-50 days led to the generation of antibodies that reacted against protein-acetaldehyde conjugates, suggesting that such adducts are formed in vivo and can act as neoantigens. Antibodies against acetaldehyde adducts should be of value in the identification of alcohol consumption and in the study of the biology of the adducts in relation to organ pathology.
Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin.
Authors: Schlechter N L , Russell S M , Spencer E M , Nicoll C S
Published: May 2006

We have used a catheterization system that permits chronic infusion into the arterial supply of one hindlimb of rats to study the direct effects of rat growth hormone and human somatomedin C on growth of the tibial epiphyseal cartilage plate in hypophysectomized rats. Rat growth hormone (0.4 microgram per day) or human somatomedin C (0.25, 1, or 4 micrograms per day) stimulated growth of the epiphyseal plate of the infused limb but not of that of the contralateral noninfused limb. The somatomedin C had a dose-related effect. Rabbit antiserum to human somatomedin C, but not normal rabbit serum, completely abolished the direct growth effect of the rat growth hormone when it was co-infused with the hormone. These results support the concept that growth hormone stimulates long bone growth by inducing local production of somatomedin, which in turn stimulates cell proliferation in an autocrine or paracrine fashion. However, they do not exclude the possibility that serum somatomedin may also play a role.
Histidine-rich domain of the knob protein of the human malaria parasite Plasmodium falciparum.
Authors: Kilejian A , Sharma Y D , Karoui H , Naslund L
Published: May 2006

Membranes of erythrocytes infected with the human malaria parasite Plasmodium falciparum develop protrusions called knobs. These structures are essential for the survival of the parasite in the host, and their induction requires the synthesis of the knob protein by the parasite. We describe the isolation of a cDNA clone encoding the amino-terminal half of the knob protein. A cDNA library was constructed from RNA prepared from ring stages of a P. falciparum isolate that has retained its ability to induce knobs (knob+ phenotype). A synthetic oligonucleotide probe encoding polyhistidine was used to isolate the cDNA clone, which encodes the amino-terminal half of a polypeptide with all the known attributes of the knob protein. The gene is not transcribed in variants that do not synthesize the knob protein and thereby cannot induce knobs (knob- phenotype). The apparent lack of transcription in knob- variants is due to different mechanisms: although the gene is present in one knob- isolate, it has been deleted in a cloned knob- variant. The primary structure of the polypeptide deduced from a partial sequence of the cDNA is distinctly different from other malarial histidine-rich polypeptides. The amino-terminal sequence shows the characteristic features of a signal peptide. This is followed by a histidine-rich domain and a subsequent region which contains one histidine. Peptide map analysis of the knob protein is consistent with the structural features deduced from the sequence analysis of the cDNA.
Characterization of cDNA coding for human factor XIIIa.
Authors: Grundmann U , Amann E , Zettlmeissl G , Küpper H A
Published: May 2006

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor XIII complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XIIIa, were used as hybridization probes. Of 0.36 X 10(6) independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XIIIa-specific sequences. The total length of the isolated factor XIIIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.
Nitrate reductase from squash: cDNA cloning and nitrate regulation
Authors: Crawford Nigel M. , Campbell Wilbur H. , Davis Ronald W.
Published: May 2006

The assimilation of nitrate in plants involves the reduction of nitrate to ammonia in two steps. The first step requires nitrate reductase, a nitrate-inducible enzyme. When seedlings of squash (
Cucurbita maxima
L.) were treated with nitrate, both nitrate reductase activity and protein were induced in the cotyledons. Poly(A)
+
RNA was prepared from cotyledons of nitrate-treated seedlings and was used to construct a λgt11 cDNA library. Using antibodies from mice immunized against purified nitrate reductase from squash, a recombinant λ phage was isolated that encoded part of the nitrate reductase mRNA. The antigens produced by the recombinant phage were used to affinity purify anti-nitrate reductase antibody from ascites fluid of immunized mice. The purified antibody bound to nitrate reductase protein on immunoblots and immunoprecipitated the enzyme from squash protein extracts. The cDNA insert (1.2 kilobases) hybridized to a 3.2-kilobase RNA that was 120-fold more abundant in nitrate-induced cotyledons compared with the uninduced tissue.
Requirement of multiple copies of a 21-nucleotide sequence in the U3 regions of human T-cell leukemia virus type I and type II long terminal repeats for trans-acting activation of transcription.
Authors: Shimotohno K , Takano M , Teruuchi T , Miwa M
Published: May 2006

The cis-acting regulatory sequence of transcription from long terminal repeats (LTRs) of human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II), which is essential for action of the virally encoded trans-acting transcriptional factor(s) designated pX(s), in HTLV-I and -II was identified. Deletion of most of the U3 region of the HTLV-I LTR resulted in loss of trans-acting transcriptional activation. However, when a tandem repeat of a 21-nucleotide sequence (GAAGGCTCTGACGTCTCCCCC) that is present in the U3 region of HTLV-I and -II LTRs was inserted into the deleted U3 region of the HTLV-I LTRs, chloramphenicol acetyltransferase activity was restored. The extent of restoration of activity was proportional to the number of copies of the sequence inserted. To test the possibility that the 21-nucleotide sequence alone is necessary for trans-activation, a sequence (AGGAACTGAAA) homologous to a type-specific viral enhancer sequence and present in the U3 region of HTLV-II LTR, but not in the same region of the HTLV-I LTR, was inserted together with the 21-nucleotide sequence into the deleted U3 region of the HTLV-I LTR. However, no significant differences of the levels of activities of those LTRs compared to the LTRs with only the 21-nucleotide sequence repeats were observed.
Purification and partial characterization of prostate-derived growth factor.
Authors: Maehama S , Li D , Nanri H , Leykam J F , Deuel T F
Published: May 2006

A potent growth-promoting polypeptide, the prostate-derived growth factor (PrDGF), has been purified to apparent homogeneity from acid extracts of rat prostatic tissue using ion-exchange, reverse-phase, and gel-permeation chromatography. PrDGF migrates as a single protein-staining band in NaDodSO4/PAGE in precise correspondence to extractable PrDGF activity in nonstained NaDodSO4 gels. PrDGF is acid- and heat-stable but is sensitive to reduction or protease treatment. PrDGF is an acidic (pI 5.0) protein of approximately equal to 25 kDa in NaDodSO4/polyacrylamide gels and of approximately equal to 6-8 kDa in reduced NaDodSO4/polyacrylamide gels. PrDGF stimulates the linear incorporation of [methyl-3H]thymidine into normal rat kidney cells between 0 and 16 ng/ml. PrDGF appears to differ from other known growth factors in chemical composition and biological properties, suggesting that PrDGF is a previously undescribed growth factor.
Insertion sequence elements of
Pseudomonas savastanoi
: Nucleotide sequence and homology with
Agrobacterium tumefaciens
transfer DNA
Authors: Yamada Tetsuji , Lee Ping-Du , Kosuge Tsune
Published: May 2006

Two types of transposable elements, IS
51
and IS
52
(IS, insertion sequence), were found in
Pseudomonas syringae
subsp.
savastanoi
(
P. savastanoi
) that spontaneously insert into and inactivate
iaaM
; the insertion results in the loss of indoleacetic acid production and attenuation of virulence. The nucleotide sequences of both IS elements have sizes and structural features common to other prokaryotic IS elements; IS
51
is 1311 base pairs (bp) long and has terminal inverted repeats of 26 bp; IS
52
is 1209 bp long and has terminal inverted repeats of 10 bp with a 1 bp mismatch. In the insertion involving IS
51
, the trinucleotide sequence CAG is duplicated within
iaaM
sequences at the recombination junction; in those involving IS
52
the tetranucleotide sequences TTAG or CTAG are duplicated within
iaaM
sequences at the recombination junction. A copy of IS
51
occurs 2.5 kilobases downstream from
IaaH
. In contrast to the high copy number of IS
51
in the genome of the bacterium, only a few copies of IS
52
are present. No nucleotide sequence homology was found between IS
51
and IS
52
. However, a striking nucleotide sequence homology was found between a 531-bp region of IS
51
and a portion of the central region of transfer DNA (T-DNA) in the octopine plasmid pTi15955 from
Agrobacterium tumefaciens
. These observations, together with our earlier finding on the homology between
iaaM
and
iaaH
and between gene 1 and gene 2 of transfer DNA, further suggest that genes for indoleacetic acid production in the two systems have a common origin.
Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase.
Authors: Farr S B , D'Ari R , Touati D
Published: May 2006

Escherichia coli double mutants (sodA sodB) completely lacking superoxide dismutase (SOD) have greatly enhanced mutation rates during aerobic growth. Single mutants lacking manganese SOD (MnSOD) but possessing iron SOD (FeSOD) have a smaller increase, and single mutants lacking FeSOD but possessing MnSOD do not show such an increase. The enhancement of mutagenesis is completely dependent on the presence of oxygen, and treatments that increase the flux of superoxide radicals produce even higher levels of mutagenesis. The presence of a plasmid overproducing either form of SOD reduces the level of mutagenesis to that of wild type, showing that the O2-dependent enhancement results from a lack of SOD. The enhancement of mutagenesis is RecA-independent, and a complete lack of SOD does not induce the SOS response during aerobic growth. However, the enhanced mutagenesis in aerobically grown sodA sodB mutants is largely dependent on functional exonuclease III, suggesting that the increased flux of superoxide radicals results in DNA lesions that can be acted on by this enzyme, leading to mutations.
Expression and function of neural cell adhesion molecule during limb regeneration.
Authors: Maier C E , Watanabe M , Singer M , McQuarrie I G , Sunshine J , Rutishauser U
Published: May 2006

The neural cell adhesion molecule (NCAM) has been detected in regenerating limb bud of adult newts in addition to brain and peripheral nerves. In the regenerating tissue, NCAM was found primarily on mesenchymal cells and also in wound epidermis. Infusion of Fab fragments of antibodies to NCAM into limb buds at the early blastema stage delayed the regenerative process. Previous studies have indicated that NCAM serves as a homophilic ligand for adhesion among cells that express this molecule and, in doing so, can influence the interaction of nerves with their environment. The expression of NCAM in regenerating limb and the effects of antibody infusion are therefore consistent with the observation that limb regeneration requires interactions among axons and mesenchymal cells.
Isolation, sequence, and developmental profile of a brain-specific polypeptide, PEP-19.
Authors: Ziai R , Pan Y C , Hulmes J D , Sangameswaran L , Morgan J I
Published: May 2006

By comparing the HPLC profiles of cerebellar extracts from adult and neonatal rats, a developmentally regulated polypeptide, termed PEP-19, was identified. The concentration of PEP-19 rose from 0.1 nmol/g of cerebellum at birth to 2 nmol/g at 20 days postpartum. The polypeptide could also be detected at lower levels in olfactory bulbs of adult rats but was absent in cerebral cortex, brain stem, and all non-neural tissues examined. HPLC-purified PEP-19 contained 61 amino acids and had a molecular size of 7.6 kDa. The native polypeptide is blocked at its amino terminus but was sequenced following proteolytic and chemical fragmentation. The primary amino acid sequence was determined to be: X (S-E) R Q S A G A T N G K D K T S G D N D G Q K K V Q E E F D I D M D A P E T E R A A V A I Q S Q F R K F Q K K K A G S Q S. PEP-19 has a unique sequence, but shares some homology with several calcium binding proteins including the beta chain of S100 and intestinal calcium binding protein. This polypeptide is the primary translation product of cerebellar poly(A)+ mRNA.
Mouse thymidylate synthase messenger RNA lacks a 3' untranslated region.
Authors: Jenh C H , Deng T L , Li D W , DeWille J , Johnson L F
Published: May 2006

Analysis of the sequence of cDNA corresponding to mouse thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) mRNA revealed that the termination codon TAA was followed immediately by a poly(A) sequence. This raised the possibility that mouse thymidylate synthase mRNA lacks a 3' untranslated region. In the present study, we have further investigated this possibility. DNA corresponding to the 3' end of the thymidylate synthase gene was isolated from a genomic library. The sequence of the genomic DNA was identical to that of the cDNA in the coding region. However, the termination codon was TAG in the genomic sequence rather than TAA, and poly(A) was not present in the genomic DNA. Sequences flanking the site of poly(A) addition were in good agreement with polyadenylylation consensus sequences. S1 nuclease analysis revealed that approximately 80% of the thymidylate synthase mRNA molecules were polyadenylylated at the termination codon. A secondary polyadenylylation site was detected 190-200 nucleotides downstream of the primary site. We conclude that the major species of mouse thymidylate synthase mRNA lacks a 3' untranslated region and that the final A of the termination codon is added by poly(A) polymerase. It appears that a 3' untranslated region is not essential for the accumulation or translation of this mRNA.
Differential sensitivity of two functions of the insulin receptor to the associated proteolysis: kinase action and hormone binding.
Authors: Kathuria S , Hartman S , Grunfeld C , Ramachandran J , Fujita-Yamaguchi Y
Published: May 2006

Since we observed that after purification the receptor kinase activity is rapidly lost under conditions where insulin binding function seems to be preserved, we have studied the cause(s) of receptor kinase inactivation. Highly purified placental insulin receptor preparations were analyzed by NaDodSO4/PAGE followed by silver staining or immunostaining using domain-specific antibodies raised against synthetic peptides corresponding to the amino acid sequences of the beta subunit. These studies revealed the intact 90-kDa beta subunit is degraded first to an 88-kDa form and then to a 50-kDa beta 1-subunit form by proteolysis even after purification when stored at 4 degrees C. The 88-kDa beta subunit, which lacks the carboxyl-terminal approximately equal to 2-kDa portion exhibits almost no autophosphorylation activity, nor does insulin stimulate autophosphorylation. The loss of kinase activity as measured by phosphorylation of the src-related peptide is correlated with the loss of the intact 90-kDa beta subunit. Degradation of the beta subunit to the 50-kDa form seems to be facilitated by the removal of the approximately equal to 2-kDa peptide. Present studies thus suggest that only the intact form of the beta subunit has full kinase activity in an insulin-dependent manner and that other forms, such as the 88-kDa beta subunit show little kinase activity. The inactivation appears to arise from a conformational change of the 90-kDa form, which makes it susceptible to proteolysis at the carboxyl-terminal end. These results imply that the carboxyl-terminal of the beta subunit is important for the manifestation of the tyrosine kinase activity of the insulin receptor.
Expression of major histocompatibility complex class I antigens as a strategy for the potentiation of immune recognition of tumor cells.
Authors: Tanaka K , Hayashi H , Hamada C , Khoury G , Jay G
Published: May 2006

Like many primary tumors, human adenovirus type 12 (Ad12)-transformed mouse cells express greatly reduced levels of the major histocompatibility complex (MHC) class I antigens and are highly tumorigenic in immunocompetent hosts. Expression of a transfected class I gene by these cells can abrogate their tumorigenicity. Both the K and the L class I genes can suppress the malignant phenotype. Previous studies showed that interferon can induce class I gene expression in certain Ad12-transformed cells and can suppress their tumorigenic phenotype. We now demonstrate that preimmunization of mice with a nontumorigenic dose of interferon-treated Ad12-transformed tumor cells can afford protection against a subsequent challenge by a tumorigenic dose of untreated Ad12-transformed tumor cells. Similar immunity can also be induced by using cells transfected with the K gene, and the observed protection appears specific to Ad12-transformed cells. Significant protection can be achieved even if immunization is provided subsequent to the tumor challenge. Since increasing numbers of human tumors have been found to have reduced levels of MHC class I antigens, the prospect of therapy by immunization with the parental tumor cells that have been manipulated to induce class I gene expression offers an attractive experimental model.
Charge movement and depolarization-contraction coupling in arthropod vs. vertebrate skeletal muscle.
Authors: Scheuer T , Gilly W F
Published: May 2006

Voltage-dependent charge movement has been characterized in arthropod skeletal muscle. Charge movement in scorpion (Centuroides sculpturatus) muscle is distinguishable from that in vertebrate skeletal muscle by criteria of kinetics, voltage dependence, and pharmacology. The function of scorpion charge movement is gating of calcium channels in the sarcolemma, and depolarization-contraction coupling relies on calcium influx through these channels.
Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.
Authors: McPhaul M , Berg P
Published: May 2006

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes.
Identification of a domain within the phosphoprotein of vesicular stomatitis virus that is essential for transcription in vitro.
Authors: Gill D S , Chattopadhyay D , Banerjee A K
Published: May 2006

A full-length cDNA copy of the phosphoprotein (NS) mRNA of vesicular stomatitis virus (New Jersey serotype) was inserted into pGEM4 vector downstream of the promoter for bacteriophage SP6 RNA polymerase. Transcription of the cDNA in vitro resulted in the synthesis of NS mRNA, which was subsequently translated into NS protein in a cell-free rabbit reticulocyte system. The biological activity of the expressed NS protein was demonstrated by in vitro synthesis of mRNA by transcription-reconstitution with purified viral L protein and N-RNA template. Deletion mapping of the NS gene defined a specific domain between amino acid residues 213 and 247, which was essential for in vitro transcription. Removal of the COOH-terminal 21 amino acids, on the other hand, did not have a significant effect on transcription. This domain appears to be involved in efficient binding of NS protein to the N protein-RNA template.
Regulated production of mu m and mu s mRNA requires linkage of the poly(A) addition sites and is dependent on the length of the mu s-mu m intron.
Authors: Peterson M L , Perry R P
Published: May 2006

mRNAs encoding the membrane-associated (mu m) and secreted (mu s) forms of mu heavy chain are derived from transcripts of the same immunoglobulin gene by differential RNA processing. To help elucidate the mechanism that regulates the production of these two mu mRNAs during the course of B-lymphoid maturation, we produced a series of specifically modified mu-chain genes and studied their expression when transfected into cells representing either early or late developmental stages. We have established that proper regulation depends on linkage of the mu s and mu m poly(A) addition sites and the length of the mu s-mu m intron. Deletion of an 800 to 900-nucleotide segment from the central region of this intron abolishes regulation; replacement of this segment with miscellaneous DNA sequences restores it. From these results we propose a model in which regulation is principally achieved by competition between cleavage/polyadenylylation of the mu s site and splicing of the C mu 4 and mu m exons.
Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.
Authors: Cleghon V G , Klessig D F
Published: May 2006

The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-terminal domain of DBP binds RNA in vitro and was found to be associated with polyadenylylated RNA in vivo. This is the same domain that interacts with DNA. However, the differences in sensitivity of DBP to trypsin when bound to RNA versus DNA suggest that RNA and DNA either bind at different sites within this domain or induce different conformational changes within the protein.
Molecular basis for two different affinity states of the interleukin 2 receptor: affinity conversion model.
Authors: Kondo S , Shimizu A , Saito Y , Kinoshita M , Honjo T
Published: May 2006

Two affinity species of the interleukin 2 (IL-2) receptor are different states of a single receptor molecule. We assumed that a binary complex between the IL-2 receptor and another lymphocyte-specific protein would constitute the high-affinity receptor. To test this assumption, we counted the numbers of IL-2 receptors with high and low affinity in a murine T-cell line CT/hR-1 that expresses not only murine but also human receptors by cDNA transfection. We found that human high-affinity receptors disappeared when the murine high-affinity receptors were already occupied by the ligand. The results were incompatible with a fixed number of human and murine receptors with high affinity in CT/hR-1 cells. We suggest that the high-affinity state of the IL-2 receptor is a ternary complex of IL-2, the IL-2 receptor, and a postulated "converter" protein, which is fewer in number than the receptors. The converter would be unable to form a complex with the IL-2 receptor unless IL-2 was already bound to it. The ligand binding to the receptor would cause a conformational change in the receptor, increasing its affinity to the converter. Ternary complex formation would, in turn, change the apparent affinity of the receptor to the ligand from low to high by reduction of the dissociation constant.
Bioluminescence in soybean root nodules: Demonstration of a general approach to assay gene expression
in vivo
by using bacterial luciferase
Authors: Legocki Roman P. , Legocki Misuk , Baldwin Thomas O. , Szalay Aladar A.
Published: May 2006

Two plasmid vectors pFIT001 and pPALE001, containing
luxAB
genes encoding bacterial luciferase [alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] from
Vibrio harveyi
, have been constructed.
Escherichia coli
carrying derivatives of pFIT001 with DNA inserts in the unique
Eco
RI site located in
luxB
form “dark” colonies that can be readily distinguished from the bioluminescent or “bright” colonies. In contrast, promoterless pPALE001 is used as a promoter-search vector based on bioluminescence. The control and regulation of gene expression can be analyzed
in vivo
using promoter-
luxAB
fusions by a variety of simple methods, including a technique called “luxdot.” As an example, we have introduced nitrogenase
nifD
and
nifH
promoter-
luxAB
fusions into the
Bradyrhizobium japonicum
chromosome and shown symbiotically regulated bioluminescence in soybean root nodules.
B. japonicum
transconjugants containing a single copy per genome of the
nif
promoter-controlled luciferase structural genes did not produce light in free-living cultures, but the same transconjugants did express bioluminescence in root nodules that was strong enough to be detected by the naked eye.
Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-related peptides in the rat adrenal medulla.
Authors: Kanamatsu T , Unsworth C D , Diliberto E J , Viveros O H , Hong J S
Published: May 2006

The effect of reflex splanchnic nerve stimulation on proenkephalin A biosynthesis was investigated in the rat adrenal medulla. Tissue levels of native [Met5]enkephalin-like immunoreactivity (IR) (measured by direct RIA of tissue extracts), cryptic [Met5]enkephalin-like IR (calculated as the increase in [Met5]enkephalin-like IR detected in tissue extracts after sequential digestion with trypsin and carboxypeptidase B), and proenkephalin A mRNA were determined in adrenal medulla from rats sacrificed at various times after a period of insulin-induced hypoglycemia. Two hours of insulin hypoglycemia, which produced intense reflex stimulation of the splanchnic nerves as evidenced by a 55% decrease in the adrenal medulla catecholamine levels, resulted in a 3-fold increase in proenkephalin A mRNA levels in this tissue. The proenkephalin A mRNA levels reached a maximum 15-fold increase over control values 24 hr after this period of hypoglycemic stress and then gradually declined with an approximate half-life of 4 days. Native and cryptic [Met5]enkephalin-like IR had increased 9-fold and 12-fold, respectively, 24 hr after this period of hypoglycemia, and both demonstrated maximum increases of 130-fold and 50-fold, respectively, after 96 hr. Combined pretreatment (i.p. administration) with the ganglionic and muscarinic blocking agents chlorisondamine (5 mg/kg of body weight) and atropine (1 mg/kg) blocked the increase in levels of proenkephalin A mRNA seen in the rat adrenal medulla following insulin hypoglycemia. These data indicate that reflex splanchnic nerve discharge stimulates proenkephalin biosynthesis, probably at the level of gene expression.
Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.
Authors: Neumann D , Barchan D , Fridkin M , Fuchs S
Published: May 2006

A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the tryptophan residue is replaced by serine, supports this hypothesis.
Molecular analysis of cDNA clones and the corresponding genomic coding sequences of the Drosophila dunce+ gene, the structural gene for cAMP phosphodiesterase.
Authors: Chen C N , Denome S , Davis R L
Published: May 2006

We have isolated and sequenced cDNA clones representing portions of the polyadenylylated transcripts of the dunce+ gene. These define an open reading frame of 1086 bases and some of the 5'- and 3'-untranslated regions of the transcripts. The deduced amino acid sequence is strikingly homologous to the amino acid sequence of a Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase isolated from bovine brain and more weakly related to the predicted amino acid sequence of a yeast cAMP phosphodiesterase. These homologies, together with prior genetic and biochemical studies, provide unambiguous evidence that dunce+ codes for a phosphodiesterase. In addition, the dunce+ gene product shares a seven-amino acid sequence with a regulatory subunit of cAMP-dependent protein kinase that is predicted to be part of the cAMP binding site. We also identify a weak homology between a region of the dunce+ gene product and the egg-laying hormone precursor of Aplysia californica. The open reading frame is divided in the genome by four introns.
Overlapping cDNA clones define the complete coding region for the P210c-abl gene product associated with chronic myelogenous leukemia cells containing the Philadelphia chromosome.
Authors: Mes-Masson A M , McLaughlin J , Daley G Q , Paskind M , Witte O N
Published: May 2006

The Philadelphia chromosome, observed in greater than 90% of patients with chronic myelogenous leukemia, results from a reciprocal translocation between chromosomes 9 and 22. The translocation breakpoint on chromosome 9 occurs near the ABL gene and correlates with the production of a chronic myelogenous leukemia-specific 8.5-kilobase ABL-related mRNA species accompanied by a structurally altered ABL protein (P210c-abl). The N-terminal sequence of the protein is derived from the BCR gene on chromosome 22. We have isolated overlapping cDNA clones from the K-562 cell line corresponding to approximately 8.5 kilobases of mRNA and have sequenced 2550 nucleotides at the 5' end. Our results indicate that the 5' end of the 8.5-kilobase mRNA consists of greater than 400 nucleotides of noncoding sequence that are greater than 80% G + C rich. Based on our sequence analysis, we propose that initiation of translation occurs at nucleotide 471, such that the initial 927 amino acids of P210c-abl are derived from BCR sequences. Our cDNA clones thus define the complete coding sequences for the P210c-abl gene product.
Isolation of a thermostable enzyme variant by cloning and selection in a thermophile.
Authors: Liao H , McKenzie T , Hageman R
Published: May 2006

We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47 degrees C but not at 55 degrees C and above. Variants that were kanamycin resistant at 63 degrees C were obtained by selection of spontaneous mutants, by passage of a shuttle plasmid through the Escherichia coli mutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the first variant by selecting for kanamycin resistance at 70 degrees C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.
Stimulation of aortic smooth muscle cell mitogenesis by serotonin.
Authors: Nemecek G M , Coughlin S R , Handley D A , Moskowitz M A
Published: May 2006

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 microM serotonin with increased incorporation of [3H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 microM. At a concentration of 1 microM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approximately 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.
Identification of functional regions on the I-Ab molecule by site-directed mutagenesis.
Authors: Cohn L E , Glimcher L H , Waldmann R A , Smith J A , Ben-Nun A , Seidman J G , Choi E
Published: May 2006

Functional analysis of mutant class II major histocompatibility complex molecules has begun to identify regions important for antibody binding and for T-cell activation. By using in vitro mutagenesis directed at the beta 1 domain of the Ab beta gene we have constructed three structurally distinct mutant Ab beta genes. Each of these genes, as well as the wild-type Ab beta gene, was cotransfected together with the wild-type Ab alpha gene into the Ia-negative B-lymphoma cell line M12.C3. Transfection resulted in the successful synthesis and cell surface expression of three mutant class II antigens that showed serological and functional alterations as compared to the I-Ab antigens from the M12.C3 cell transfected with the wild-type gene. The variable patterns of both I-Ab-specific monoclonal antibody binding and activation of I-Ab-specific T-cell hybridomas show that the mutations result in the loss of structural epitopes required for both monoclonal antibody binding and for T-cell recognition. The data suggest that there are multiple sites on a single Ia molecule that are recognized by T helper cells and also that the tertiary conformation of the Ia molecule can be critical in the formation of such sites.
Murine T-cell receptor mutants with deletions of beta-chain variable region genes.
Authors: Behlke M A , Chou H S , Huppi K , Loh D Y
Published: May 2006

Genomic Southern blots of DNA from eight strains of mice were examined for restriction fragment length polymorphisms in their loci encoding the variable region of the T-cell receptor beta chain (V beta), using 16 different V beta-specific probes. Mouse strains BALB/c, C57BL/6, C3H, and PL were identical, while strains SJL, C57BR, C57L, and SWR shared several polymorphisms with respect to the other four strains. In addition, SJL, C57L, C57BR, and SWR DNAs were missing 50% of the hybridizing bands visualized in BALB/c DNA. A cDNA library from concanavalin A-stimulated SJL spleen blasts was constructed and examined for V beta gene usage. Ten genes were found to account for all V beta-containing clones isolated, including three newly identified V beta genes. All 10 of these genes were found to be present in BALB/c mice. We conclude that SJL, C57L, C57BR, and SWR mice represent V beta deletion mutants of the BALB/c genotype.
Genetic analysis of the fragile-X mental retardation syndrome with two flanking polymorphic DNA markers.
Authors: Oberlé I , Heilig R , Moisan J P , Kloepfer C , Mattéi G M , Mattéi J F , Boué J , Froster-Iskenius U , Jacobs P A , Lathrop G M
Published: May 2006

The fragile-X mental retardation syndrome, one of the most prevalent chromosome X-linked diseases (approximately equal to 1 of 2000 newborn males), is characterized by the presence in affected males and in a portion of carrier females of a fragile site at chromosomes band Xq27. We have performed a linkage analysis in 16 families between the locus for the fragile-X syndrome, FRAXQ27, and two polymorphic DNA markers that correspond to the anonymous probe St14 and to the coagulation factor IX gene F9. Our results indicate that the order of loci is centromere-F9-FRAXQ27-St14-Xqter. The estimate of the recombination fraction for the linkage F9-FRAXQ27 is 0.12 (90% confidence limits: 0.044-0.225) and 0.10 for FRAXQ27-St14 (90% confidence limits: 0.040-0.185). Recombination between St14 and F9 does not appear to be significantly different in normal and fragile-X families. The two flanking probes were used for diagnosis of the carrier state and for detection of transmission of the disease through phenotypically normal males. They should also allow first-trimester diagnosis with a reliability of about 98% in 40% of the families. Used in conjunction with the cytogenetic analysis, the segregation studies with both probes should improve the genetic counseling for the fragile-X syndrome and should be useful for the formal genetic analysis of this unique disease.
Elevated erythrocyte adenosine deaminase activity in patients with acquired immunodeficiency syndrome.
Authors: Cowan M J , Brady R O , Widder K J
Published: May 2006

Acquired immunodeficiency syndrome (AIDS) is an often fatal disease caused by a retrovirus frequently resulting in malignancy and/or opportunistic infection. Because the immune deficiency in AIDS is similar to that in some purine enzyme deficiencies, we measured erythrocyte adenosine deaminase (ADA) and purine nucleoside phosphorylase activities in patients with AIDS, heterosexual controls, and a high-risk asymptomatic population. We found that erythrocyte ADA activity was significantly elevated in patients with AIDS (40 +/- 11 nmol/mg of hemoglobin per hr, mean +/- SD) relative to heterosexual controls (25 +/- 10, P less than 0.001). We also measured ADA activity in a group of individuals at high risk for AIDS and found that approximately half had significantly elevated ADA activities (45 +/- 4, P less than 0.002) that correlated with the presence of antibody to the lymphadenopathy retrovirus. Purine nucleoside phosphorylase activity was relatively normal in patients with AIDS as well as in individuals at risk for AIDS. Increased ADA appears to be a diagnostic marker of AIDS and may be useful in conjunction with antibody to the AIDS-related retrovirus in detecting the presence of infection in asymptomatic high-risk individuals. These data also suggest that, in addition to the lymphocyte, the erythroid cell line may also be infected by the AIDS-related retrovirus.