The three-dimensional structure of BirA, the repressor of the Escherichia col biotin biosynthetic operon, has been determined by x-ray crystallography and refined to a crystallographic residual of 19.0% at 2.3-A resolution. BirA is a sequence-specific DNA-binding protein that also catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers the biotin moiety to other proteins. The level of biotin biosynthetic enzymes in the cell is controlled by the amount of biotinyl-5'-adenylate, which is the BirA corepressor. The structure provides an example of a transcription factor that is also an enzyme. The structure ofBirA is highly asymmetric and consists of three domains. The N-terminal domain is mostly a-helical, contains a helix-turn-helix DNA-binding motif, and is loosely connected to the remainder of the molecule. The central domain consists of a seven-stranded mixed 13-sheet with a-helices covering one face. The other side of the sheet is largely solventexposed and contains the active site. The C-terminal domain comprises a six-stranded, antiparallel 13-sheet sandwich. The location of biotin binding is consistent with mutations that affect enzymatic activity. A nearby loop has a sequence that has been associated with phosphate binding in other proteins. It is inferred that ATP binds in this region, adjacent to the biotin. It is proposed that the binding of corepressor to monomeric BirA may promote DNA binding by facilitating the formation of a multimeric BirA-corepressor-DNA complex. The structural details of this complex remain an open question, however.The biotin operon repressor, BirA, is a 33.5-kDa protein that regulates transcription of the Escherichia coli biotin operon (1-3). BirA is bifunctional, serving both as the biotin (vitamin H)-activating enzyme and as a transcriptional regulator. It catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers biotin to a specific lysine residue on the biotin carboxyl carrier protein, a subunit of acetyl-CoA carboxylase (4,5). If all the biotin-accepting proteins in the cell have been biotinylated, the BirA-biotinyl-5'-AMP complex accumulates and binds to the 40-base-pair bio operator, repressing transcription of the biotin biosynthetic genes (4, 6-8). Thus BirA synthesizes its own corepressor, a unique property among known DNA-binding proteins. BirA represses transcription when biotinyl-5'-AMP is bound to the enzyme, suggesting that binding of corepressor helps form the BirA-DNA complex. Structure DeterminationCrystals of BirA were grown as described (9) and equilibrated with 2.05 M phosphate, pH 6.5/5% (vol/vol) glycerol. Native and derivative data sets were collected by using film or a Xuong-Hamlin (10) area detector ( Table 1). Inclusion of anomalous data in cross-phased difference Fourier maps showed the space group to be P43212 rather than its enantiomorph.Refined heavy-atom parameters were employed to compute multiple isomorphous replacement phases to 3.0-A resolution. The mean figure of merit, including an...

Abstract. We cloned a novel ankyfin, Ank3, from mouse kidney cDNA. The full-length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin-binding" domain; and a 56 kD, COOH-terminal

Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs. Stepwise assembly of the gene segments seems to occur by replacement of the intervening DNA between the 5' and 3' constant-region genes. Here we report that lamprey (Lethenteron japonicum) assemble their VLR genes by a process involving 'copy choice'. Regions of short homology seemed to prime copying of donor LRR-encoding sequences into the recipient gene. Those LRR-encoding germline sequences were abundant and shared extensive sequence homologies. Such genomic organization permits initiation of copying anywhere in an LRR-encoding module for the generation of various hybrid LRRs. Thus, a vast repertoire of recombinant VLR genes could be generated not only by copying of various LRR segments in diverse combinations but also by the use of multiple sites in an LRR gene segment for priming.

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Caveolae are vesicular organelles that represent an appendage of the plasma membrane. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Caveolin has been proposed to function as a plasma membrane scaffolding protein to organize and concentrate signaling molecules within caveolae, including heterotrimeric G proteins (␣ and ␤␥ subunits). In this regard, caveolin interacts directly with G ␣ subunits and can functionally regulate their activity. To date, three cDNAs encoding four subtypes of caveolin have been described in vertebrates. However, evidence for the existence of caveolin proteins in less complex organisms has been lacking. Here, we report the identification, cDNA sequence and genomic organization of the first invertebrate caveolin gene, Cav ce (for caveolin from Caenorhabditis elegans). The Cav ce gene, located on chromosome IV, consists of two exons interrupted by a 125-nucleotide intron sequence. The region of Cav ce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cav ce. This suggests that mammalian caveolins may have evolved from the second exon of Cav ce. Cav ce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. Remarkably, the invertebrate Cav ce protein behaves like mammalian caveolins: (i) Cav ce forms a high molecular mass oligomer, (ii) assumes a cytoplasmic membrane orientation, and (iii) interacts with G proteins. A 20-residue peptide encoding the predicted G protein binding region of Cav ce possesses "GDP dissociation inhibitor-like activity" with the same potency as described earlier for mammalian caveolin-1. Thus, caveolin appears to be structurally and functionally conserved from worms to man. In addition, we find that there are at least two caveolin-related genes expressed in C. elegans, defining an invertebrate caveolin gene family. These results establish the nematode C. elegans as an invertebrate model system to study caveolae and caveolin in vivo. Caveolae are vesicular organelles that represent a subdivision of the plasma membrane (1, 2). Although they are found in most cell types, caveolae are most numerous in adipocytes, endothelial cells, fibroblasts, and muscle cells (smooth, skeletal, and cardiac). In transmission electron micrographs they can be distinguished by their characteristic shape: ϳ50-100-nm vesicles located at or near the plasma membrane (3, 4). Functionally, caveolae have been implicated in a variety of signal transduction related events, including signaling from G protein-coupled receptors and growth factor receptors (5-7). Caveolin, a 21-24-kDa protein, is an integral membrane component of caveolae (8-12). Caveolin co-purifies with lipid modified signaling molecules, such as G proteins (␣ and ␤␥ subunits), Src-family tyrosine kinases, and H-Ras (see Refs. 6, 9, and 13-18 and references cited within). These molecules appear to be tightly associated as a discrete complex with caveolin as shown using a polyhistidine-tagg...

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixthand seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequencespecific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.V(D)J joining is a site-specific recombination process that plays a crucial role in the activation and diversification of antigen receptor genes (44). Joining occurs between two pairs of recombination signal sequences (RSSs): heptamer (CACA GTG) and nonamer (ACAAAAACC) (22,32). Furthermore, the spacer separating the heptamer and the nonamer is either 12 or 23 bp in length, and recombination takes place between two RSSs in which one contains a 12-bp spacer (12-RSS), and the other contains a 23-bp spacer (23-RSS) (8,33,34); this is the so-called 12/23 rule. It has been shown that just two pairs of the heptamer and the nonamer are sufficient for V(D)J type recombination if the 12/23 rule is satisfied (3).V(D)J type recombination consists of two major processes: site-specific cleavage and ligation of cleaved ends. The former process includes specific recognition of the RSS by DNAbinding components of the recombinase, synaptic complex formation between the two RSSs satisfying the 12/23 rule, and site-specific cleavage of RSSs adjacent to the heptamer (29, 37). The latter process is known to be mediated by DNA repair mechanisms, including DNA-dependent protein kinase, (4,17,19), the Ku protein complex (43, 47), XRCC4 (21), and DNA ligases (13,28). During the process of recombination, nucleotide deletion and addition occur at coding ends. Terminal deoxynucleotidyl transferase is responsible for the insertion of non-germ line nucleotides (11, 18).Two recombination-activating genes, rag-1 and rag-2, were isolated by their abilities to activate V(D)J type recombination in a fibroblast cell line (26, 36). It was not clear for many years what roles RAG proteins played in the process of V(D)J recombination. The recent demonstration of in vitro RSS cleavage (45) provided a more convincing argument that the RAG proteins were indeed major com...

Abstract. Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and AOI3 ankyrin (x<4700 aa, ~<517 kD). In addition to the major set of •6 kb alternatively spliced transcripts, minor transcripts were observed at -,3, 5, 7, and 14 kb. Evidence is provided that mutations in the *14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.TrIOUGH the molecular basis of neural development has been the object of intense study in recent years, the detailed mechanisms of axon guidance remain unknown (for general reviews see Dodd and Jessell, 1988;Jessell, 1988;Takeichi, 1988;Sanes, 1989;Takeichi, 1991;Rathjen et al., 1992;Gumbiner, 1993; for C. elegans reviews see Hedgecock et al., 1987;Wadsworth and Hedgecock, 1992).Mutations in the unc-44 gene affect the direction of axonal outgrowth for many axons (Hedgecock et al., 1985;Siddiqui, 1990;Siddiqui and Culotti, 1991;Mclntire et al., 1992), including the postdeirid (PDE) ~ axon, which normally extends from the postdeirid sensillum on the lateral surface of the nematode to the ventral nerve cord (White et al., 1986). In unc-44 mutants, the initial direction of PDE axon outgrowth along the basement membrane is apparently random, and the misdirected PDE axon fasciculates with inappropriate partners (Hedgecock et al., 1985 The discovery that the C. elegans unc-6 gene encodes a laminin B chain-related product provided evidence that directed axonal outgrowth and cell migration require interactions with the extracellular matrix (Hedgecock et al., 1990), and that these interactions use laminin or related proteins in both invertebrates and vertebrates (Jessell, 1988;Sanes, 1989;Hedgecock et al., 1990;Serafini et al., 1994). The product of the unc-5 gene, which affects dorsalward cell migrations and axon outgrowth, has been proposed to be a cell surface protein which may interact with the extracellular matrix (Leung-Hagesteijn et al., 1992). Thus, it was likely that other mutations affecting axonal outgrowth and guidance were defects in cytoskeletal or extracellular matrix structures. The actin/a-actinin framework of growth cone filopodia or the spectrin/ankyrin network underlying the cytoplasmic surface of the plasma membrane could be the targets for mutations affecting axon outgrowth and growth cone adhesion. In this study, we have discovered that the wild-type unc-44 gene, which is required for proper axonal guidance, encodes a series of putative ankyrin-related proteins.Ankyrin (or bands 2.1 and 2.2) has been most thoroughly studied in erythrocyte "ghost...

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