Glutamic acid decarboxylase expression in islets and brain.
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Brain Chemistry↗
Biomedical subjects
R Giorda
Publications and source records attributed to R Giorda.
Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells.
NK cells are a subpopulation of large granular lymphocytes. They are able to recognize and lyse a wide variety of virally infected or neoplastic target cells without previous sensitization or MHC restriction. The molecules involved in target recognition and subsequent triggering of the killing process are still undefined. Recently, a 30-kDa protein highly expressed on rat NK cells and capable of mediating transmembrane signaling was identified and the gene coding for it cloned and sequenced. To better understand the role of this protein in NK cell-mediated cytotoxicity, we cloned its mouse homologue by cross-hybridization of the rat gene to a cDNA library generated from highly purified mouse lymphokine-activated NK cells. Three messages, differing in size and sequence and encoded by different genes, are specifically cotranscribed in mouse NK cells. The protein products of this gene family express the lectin-like motif characteristic of type II transmembrane molecules. Both the rat and mouse proteins have conserved tyrosine and serine residues in their cytoplasmatic portion that are potential phosphorylation sites. They also share a sequence that could be the binding site of the P56lck tyrosine kinase. These observations are consistent with the signaling function hypothesized for these proteins.
Amino Acid Sequence↗
Dictyostelium discoideum gene family contains a long internal amino acid repeat.
Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.
Amino Acid Sequence↗
Full length beta chain cDNAs of DQw9 and DQw8 molecules encode proteins that differ only at amino acid 57.
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Amino Acid Sequence↗
NKR-P1, a signal transduction molecule on natural killer cells.
Natural killer (NK) cells are a subpopulation of large granular lymphocytes characterized by densely staining azurophilic granules. NK cells are able to recognize and lyse various virally infected or neoplastic target cells without previous sensitization or major histocompatibility complex restriction. A 60-kD disulfide-linked dimer, highly expressed on NK cells, was found capable of mediating transmembrane signaling. The gene encoding this signal transduction molecule was cloned and its nucleotide sequence determined. The encoded protein showed significant homology with a number of lectin-related membrane proteins that share receptor characteristics. This protein may function as a receptor able to selectively trigger NK cell activity.
Amino Acid Sequence↗
A shared internal threonine-glutamic acid-threonine-proline repeat defines a family of Dictyostelium discoideum spore germination specific proteins.
A cDNA denoted pRK270 hybridizes to two mRNA species in RNA blots. The mRNAs specific to this clone are not expressed during vegetative growth and multicellular development. They are, however, found predominantly during early stages of spore germination, suggesting that their synthesis is rapidly and coordinately turned on during germination. Two different cDNAs named 270-6 and 270-11 were isolated, representing the two mRNAs. DNA blot analysis shows that 270 is a multigene family. Four genes were isolated from Dictyostelium genomic libraries and sequenced. The putative proteins coded for by these genes are about 51,000, 55,000, 76,000, and 100,000 Da. Two of the genes are expressed during spore germination while transcripts for the other two are not present during spore germination, vegetative growth, or the stages of multicellular development studied. The cDNAs and genes code for deduced proteins that possess a very unusual internal amino acid repeat comprised of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The 270-6 protein shows excellent identity with avocado (Persea americana) cellulase, indicating that it may function as an endo-(1,4)-beta-D-glucanase.
Amino Acid Sequence↗
Isolation and characterization of a cDNA that encodes the core protein of the cytolytic granule proteoglycan in rat natural killer cells.
In this report, we describe the transcription of messenger ribonucleic acid (mRNA) specific for the core protein of chondroitin sulfate proteoglycan (CSPG), the entire sequence of the message for CSPG core protein and the presence of CSPG in the granules of a highly purified population of recombinant interleukin-2 (rIL-2)-stimulated rat natural killer (NK), i.e. adherent lymphokine-activated Killer (A-LAK), cells. The presence of CSPG in A-LAK cell granules was demonstrated by a variety of biochemical and immunologic methods. Further, we have demonstrated the presence of a 1.1-kilobase (kb) transcript for the core protein of CSPG by Northern blot analysis using a specific probe (pPG6) derived from a rat yolk sac tumor cell line (L-2). Three cell types that contained CSPG in granules produced a transcript of 1.1 kb, whereas L-2 cells, which localize CSPG on the cell surface, produced a transcript of 1.3 kb. A complementary DNA (cDNA) library was prepared from rat A-LAK cells and the gene for the CSPG core protein was cloned. From approximately 1.2 X 10(5) recombinant phages, 5 positive clones were obtained. The longest clone, PG-NK-5, was sequenced in its entirety and it was found to encode the entire sequence of the CSPG core protein. The other 4 clones were partially sequenced and were identical to PG-NK-5. Comparison of the sequence of PG-NK-5 with pPG6 indicated that they were nearly identical. However, all NK-cell-derived cDNAs contained a poly(A) tail that started 20 basepairs upstream from other published sequences for CSPG core proteins. These data represent the first description of the sequence of the core protein of CSPG contained in NK cells.
Aggrecans↗
Molecular organization of developmentally regulated Dictyostelium discoideum ubiquitin cDNAs.
Dictyostelium discoideum ubiquitin mRNAs are regulated in a complex fashion during spore germination and multicellular development. Species of mRNA of 1900, 1400, 1100, 840, 580, and 500 nucleotides (nt) are found which are expressed differentially during different stages of development. DNA blot analysis indicates that ubiquitin genes constitute a multigene family of at least six genes. cDNAs representing all the ubiquitin mRNA transcripts were isolated and sequenced. The Dictyostelium mRNAs are organized as tandem repeats of the 76 amino acid ubiquitin unit (228 nt). We isolated one cDNA containing seven of these tandem repeats, and two different five- and three-repeat cDNAs. In addition, 2 cDNAs containing a single ubiquitin repeat fused at its 3' end to an unrelated 52 and 78 amino acid extension were identified. There is a remarkable similarity in the sequences of the non-ubiquitin extensions among yeast and mammalian counterparts. The extensions are very basic, containing approximately 30% lysine/arginine. Another common feature of these proteins is the presence of a common structural motif containing cysteine residues at conserved positions, suggesting a metal binding domain that matches a consensus sequence of Xenopus transcription factor TFIIIA and other nucleic acid binding proteins. The characterization of ubiquitin cDNAs and genomic sequences in D. discoideum now makes the understanding of its developmental regulation feasible.
Amino Acid Sequence↗
Organization of a gene family developmentally regulated during Dictyostelium discoideum spore germination.
mRNA specific to cDNA clone pLK109 is present in Dictyostelium discoideum spores, increases about two- to threefold at 0.5 to 1 h during spore germination, and then rapidly decreases. The mRNA is not detectable in vegetative cells or in early multicellular development on filters, but is present late during development, approximately at the time of sporulation. 109 mRNA in spores is 700 nucleotides in length but this is processed during germination by shortening of the poly(A) tail to about 600 nucleotides at 1 to 1.5 hours. pLK109 is a member of a multigene family containing three separate genes, and we have isolated and sequenced all of them. All three sequences code for deduced proteins of 127 amino acid residues, with only a few amino acid differences among them. Gene 1 represents the "transcribed" gene, since all 33 cDNAs we isolated are identical with the cDNA pLK109 and the coding region of this gene. Other open reading frames are in close proximity to each of the 109 sequences. About 200 base-pairs 3' to the gene 1 109 sequence is an open reading frame in the opposite orientation. Gene 2 fragment contains a sequence that codes for a protein similar to trypanosome alpha-tubulin 728 base-pairs 5' to the 109 sequence. Gene 3 fragment possesses two additional putative coding regions, one 5' and another 3' to the 109 gene. There is a remarkable similarity between the 5' upstream regions of all three genes. Each possesses a normal Dictyostelium TATA box and the usual T stretch. In addition, there are many other portions of about 400 to 500 base-pairs of the 5' regions that are either identical for long stretches or very similar.
Amino Acid Sequence↗
Nucleotide sequences of Dictyostelium discoideum developmentally regulated cDNAs rich in (AAC) imply proteins that contain clusters of asparagine, glutamine, or threonine.
A Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.
Asparagine↗
Analysis of specific mRNA destabilization during Dictyostelium development.
A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.
Dictyostelium↗
Rapid detection of IDDM susceptibility with HLA-DQ beta-alleles as markers.
The allelic forms of the human leukocyte antigen (HLA)-DQ beta-chain (DQB1) have been recognized as the best markers of insulin-dependent diabetes mellitus (IDDM) susceptibility. We describe a method that allows the recognition of these DQB1 alleles without the use of either allele-specific oligonucleotide probes or radioactive material. This method determines these alleles by electrophoretically separating restriction enzyme-generated fragments from the polymerase chain-reaction-amplified second exon of the HLA-DQB1 gene, which encodes the first domain of the protein chain. This digestion method, which is simpler and more rapid than the previously adopted hybridization method, is described in detail to enable individuals at any clinical laboratory to quickly ascertain IDDM susceptibility.
Alleles↗
Characterization of genes that are developmentally regulated during Dictyostelium discoideum spore germination.
Similar to other stages in Dictyostelium development, spore germination is a particularly suitable model for studying the regulation of gene expression, because developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. Spores are constitutively dormant and must be activated to germinate. Under the proper environmental conditions, spores germinate in a highly synchronous manner to give rise to individual amoebae that can then enter the vegetative growth phase. Protein synthesis is developmentally regulated during this process. Because protein synthesis is transcriptionally controlled during spore germination, the respective genes must be developmentally transcribed, and these can be isolated and analyzed. Three cDNA clones specific for mRNA developmentally regulated during spore germination have been characterized and used as probes to study mRNA accumulation and decay during spore germination. Because we are interested in defining the sequences of developmentally regulated genes that may relate to their regulation of transcription, we have sequenced the cDNAs and have isolated and sequenced their respective genomic clones. The sequences of the three gene families, their genomic organization, and their special structural features are described.
Amino Acid Sequence↗
Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.
A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.
Amino Acid Sequence↗
mRNA stabilization controls the expression of a class of developmentally regulated genes in Dictyostelium discoideum.
During the development of Dictyostelium discoideum, several thousand new mRNA species appear in the cytoplasm after the cells have formed stable aggregates. Here we show that six of these late mRNAs, corresponding to six clones randomly chosen from a genomic library, are synthesized from the very beginning of development at a rate comparable to that observed late in development but that transcripts do not accumulate until after aggregation. The early- and late-synthesized mRNAs are identical in size and compete with each other for hybridization to the genomic clones. The early-synthesized mRNAs do not accumulate in the cytoplasm in the preaggregation stage because they are very unstable. Their stability, estimated from the kinetics of incorporation during continuous labeling with (32)P, increases by perhaps an order of magnitude in the postaggregation stage. We conclude that mRNA stabilization is the major controlling factor of the expression of these genes.
Journal Article↗
Case report: essential iris atrophy in the Williams-Beuren syndrome.
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Atrophy↗