CDD: a curated Entrez database of conserved domain alignments

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.     

A Mammalian bromodomain protein,brd4, interacts with replication factor C and inhibits progression to S phase

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.     

Molecular genetics of GM2-gangliosidosis AB variant: a novel mutation and expression in BHK cells

The GM2 activator is a hexosaminidase A-specific glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. Genetic deficiency of GM2 activator leads to a neurological disorder, an atypical form of Tay-Sachs disease (GM2 gangliosidosis variant AB). Here, we describe a G⁵⁰⁶ to C transversion (Arg¹⁶⁹ to Pro) in the mRNA of an infantile patient suffering from GM2-gangliosidosis variant AB. Using the polymerase chain reaction amplification and direct-sequencing technique, we found the patient to be homozygous for the mutation, whereas the parents were, as expected, heterozygous. BHK cells transfected with a construct of mutant cDNA gave no GM2 activator protein detectable by the Western blotting technique, whereas those transfected by a wild-type cDNA construct showed a significant level of human GM2 activator protein. The substitution of proline for the normal Arg¹⁶⁹ therefore appears to result in premature degradation of the mutant GM2 activator, either during the post-translational processing steps or after reaching the lysosome. The basis for the phenotype of GM2 gangliosidosis variant AB may therefore be either inactivation of the physiological activator function by the point mutation or instability of the mutant protein.     

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the -lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml^–1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml^–1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml^–1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml^–1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Newly discovered polyamine, 2-hydroxyspermidine, in Pseudomonas acidovorans.

A previously unknown hydroxylated polyamine has been recovered from Pseudomonas acidovorans 29. It has been identified as 2-hydroxyspermidine, N4-(3-aminopropyl)-1,4-diaminobutane-2-ol, by its chromatographic behavior, electrophoretic mobility, and reaction with metaperiodate. It can be synthesized enzymatically from 2-hydroxyputrescine by cell-free preparations from Escherichia coli or P. acidovorans 29 which contain propylamine transferase. It is interesting to note that the naturally occurring compound is the 2-hydroxyspermidine and not the 3-hydroxyspermidine, N1-(3-aminopropyl)-1,4-diaminobutane-2-ol, indicating that the propylamine transferase reacts preferentially with the amine distal to the hydroxyl group. A mixture of 2- and 3-hydroxyspermidines and hydroxyspermine was synthesized by reacting acrylonitrile with 2-hydroxyspermidine and catalytic reduction of the products with hydrogen. N-(gamma-aminopropyl)-beta-alanine, used to help identify the hydroxyspermidines, was synthesized from N-(3-aminopropyl)-3-aminopropanenitrile by hydrolysis with 10% NaOH.