A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from twodimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of twodimensional gel spots. The method was applied to a twodimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identiy.The identification of a purified protein is necessary in many areas of biochemical research. As the resolution and sensitivity of purification tools increase, the demand for protein sequencing increases. For example, a single high-resolution two-dimensional polyacrylamide gel can separate hundreds of proteins (1,2). Identification of all the resolvable proteins on a two-dimensional gel by conventional protein sequencing is a daunting task. The correlation of DNA from large-scale sequencing projects with their protein products will continue to place increasing demands upon protein sequencing.Proteins that are N-terminally blocked present an additional challenge since they cannot be directly sequenced by Edman degradation. Blockage may occur by posttranslational modification during protein synthesis or during purification. Many intracellular proteins have been reported to be N-terminally acetylated (3). In order to obtain internal sequence on a blocked protein, 50-100 pmol of material is usually required. The blocked protein is chemically or enzymatically cleaved. The peptides are then separated by HPLC and sequenced, a process which can take 3-4 days. In addition, proteins initially thought to be novel may, after purification and sequencing, be found already to exist in the protein sequence database. As a result, a significant fraction of sequencer time is spent simply identifying known proteins.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 1...

The formation of brain circuits requires molecular recognition between functionally related neurons. We report the cloning of a molecule that participates in these interactions. The limbic system-associated membrane protein (LAMP) is an immunoglobulin (Ig) superfamily member with 3 Ig domains and a glycosyl-phosphatidylinositol anchor. In the developing forebrain, lamp is expressed mostly by neurons comprising limbic-associated cortical and subcortical regions that function in cognition, emotion, memory, and learning. The unique distribution of LAMP reflects its functional specificity. LAMP-transfected cells selectively facilitate neurite outgrowth of primary limbic neurons. Most striking, administration of anti-LAMP in vivo results in abnormal growth of the mossy fiber projection from developing granule neurons in the dentate gyrus of the hippocampal formation, suggesting that LAMP is essential for proper targeting of this pathway. Rather than being a general guidance cue, LAMP likely serves as a recognition molecule for the formation of limbic connections.

Rho factor has been purified from a strain of E. coli containing the Su78 mutation in the suA gene and assayed in another strain with an amber mutation in the suA gene. The rho from the Su78 mutant strain is present in normal amounts but has altered termination function; it does not terminate transcription at some sites that are recognized effectively by the rho factor from the isogenic wild-type strain. Rho in cells with an amber mutation in the suA gene has been assayed by its RNAdependent ATPase activity. Extracts of cells of this strain have only 9% as much of this rho activity as extracts of cells of the isogenic wild-type strain. These results suggest that rho is the product of the suA gene. Since mutations in the su,4 gene are known to decrease polar effects of mutations in other genes, it is also suggested that rho factor is at least partially responsible for polar effects.Many nonsense and frameshift mutations in one gene of an operon pleiotropically reduce the level of expression of the genes in the operon that lie on the operator-distal side of the mutant gene (1). This polar effect is evident in the levels of both the messenger RNA sequences corresponding to the affected genes and the protein products (2, 3). In Escherichia coli, second-site mutations in the suA gene can partially relieve the polar effect of the original mutation without suppressing the orignal mutation (4, 5). Since these suA gene mutations are recessive to the wild-type allele (5) and since amber (nonsense) 8uA mutations have been found (6), it has been suggested that the product of the wild-type suA gene is a protein required for the full polar effect (6). In this paper we present evidence that the product of the suA gene is the rho transcription termination factor.Rho factor was first isolated and purified from E. coli K12 by Roberts, who showed that it causes specific termination of the synthesis of X RNA molecules in vitro (7). It has since been shown that rho is active in terminating transcription from a large number of natural DNA templates (8). Roberts also showed that rho is not a ribonuclease; it does not cleave or degrade large, isolated X RNA molecules even in complete RNA polymerase reaction mixtures (7). We now know that it does not catalyze the degradation of nascent RNA molecules either (9). On the other hand, when RNA molecules are present, rho does catalyze the hydrolysis of nucleoside triphosphates to nucleoside diphosphates and orthophosphate (9).Although the significance of this ATPase activity is not yet clear, it can be used for a convenient quantitative assay of rho factor. We have used this assay to identify and purify rho factor from cells with mutations in the suA gene. RESULTSThe rho proteins from a 8uA -strain isolated by Carter and Newton (Su78) (10) and an isogenic strain with the wild-type 1725 allele for the suA gene have been purified to homogeneity.The yields in both cases are the same: about 0.12 mg of protein from 20 g of cells, which is similar to yields from other cells by means of the ...

No abstract

No abstract