Mutational analysis of the Caenorhabditis elegans ankyrin gene unc-44 demonstrates that the large spliceoform is critical for neural development.

In Caenorhabditis elegans, unc-44 mutations affect axonal outgrowth and guidance, leading to locomotory defects. The wild-type unc-44 gene encodes a family of ankyrin proteins, which, in addition to the conventional ankyrins, includes a novel ankyrin isoform with an extended C-terminal domain, referred to AO13 ankyrin. Six spontaneous unc-44 mutations and their reversions were analyzed in order to localize regions critical for gene function. The q331::Tc1 and rh1013::Tc1 mutations were mapped to the portion of the gene encoding the conventional ankyrins, mn339 had an uncharacterized 2-kb insertion in the serine/threonine/glutamic acid/proline-rich (STEP) repeat block 5, st200::Tc5(variant) and rh1042::Tc1 were localized near the C-terminus, and mn259 resulted from two Tc1 insertions, one in STEP block 6 and the other near the C-terminus. Tc1 excisions in several revertants resulted either in the restoration of the wild-type sequence, or were associated with small in-frame deletions or insertions. Reversion of mn339 resulted in the net excision of 2463 bp of genomic DNA, including the region encoding parts of STEP blocks 5 and 6 and the intervening hydrophobic region. Interestingly, additional Tc1 insertions at a 5' exon/intron boundary were found in revertants of st200 and rh1042. Reversion of the st200::Tc5 mutation resulted in excision of the Tc5 element, and the insertion of two copies of Tc1 at different sites. The wild-type unc-44 gene produces multiple transcripts - shorter RNAs determined to be approximately 1, 3.2, 5, 6, and 7 kb long, and two large transcripts estimated to be 22 and 26 kb in length. The largest transcripts were affected by all unc-44 mutations and are proposed to be essential for axonal outgrowth and guidance.

The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II.

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.

Single-molecule behavior of monomeric and heteromeric kinesins.

Conventional kinesin is capable of long-range, processive movement along microtubules, a property that has been assumed to be important for its role in membrane transport. Here we have investigated whether the Caenorhabditis elegans monomeric kinesin unc104 and the sea urchin heteromeric kinesin KRP85/95, two other members of the kinesin superfamily that function in membrane transport, are also processive. Both motors were fused to green fluorescent protein, and the fusion proteins were tested for processive ability using a single-molecule fluorescence imaging microscope. Neither unc104-GFP nor KRP85/95-GFP exhibited processive movement (detection limit approximately 40 nm), although both motors were functional in multiple motor microtubule gliding assays (v = 1760 +/- 540 and 202 +/- 37 nm/s, respectively). Moreover, the ATP turnover rates (5.5 and 3.1 ATPs per motor domain per second, respectively) are too low to give rise to the observed microtubule gliding velocities, if only a single motor were driving transport with an 8 nm step per ATPase cycle. Instead, the results suggest that these motors have low duty cycles and that high processivity may not be required for efficient vesicle transport. Conventional kinesin's unusual processivity may be required for efficient transport of protein complexes that cannot carry multiple motors.

Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination.

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 sixth- and 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 sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

Identification, sequence, and expression of an invertebrate caveolin gene family from the nematode Caenorhabditis elegans. Implications for the molecular evolution of mammalian caveolin genes.

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 (alpha and betagamma subunits). In this regard, caveolin interacts directly with Galpha 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, Cavce (for caveolin from Caenorhabditis elegans). The Cavce gene, located on chromosome IV, consists of two exons interrupted by a 125-nucleotide intron sequence. The region of Cavce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cavce. This suggests that mammalian caveolins may have evolved from the second exon of Cavce. Cavce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. Remarkably, the invertebrate Cavce protein behaves like mammalian caveolins: (i) Cavce 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 Cavce 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.

Ank3 (epithelial ankyrin), a widely distributed new member of the ankyrin gene family and the major ankyrin in kidney, is expressed in alternatively spliced forms, including forms that lack the repeat domain.

We cloned a novel ankyrin, 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 "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7-kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins.

An ankyrin-related gene (unc-44) is necessary for proper axonal guidance in Caenorhabditis elegans.

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 AO13 ankyrin (< or = 4700 aa, < or = 517 kD). In addition to the major set of approximately 6 kb alternatively spliced transcripts, minor transcripts were observed at approximately 3, 5, 7, and 14 kb. Evidence is provided that mutations in the approximately 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.

Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains.

The three-dimensional structure of BirA, the repressor of the Escherichia coli 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 of BirA is highly asymmetric and consists of three domains. The N-terminal domain is mostly alpha-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 beta-sheet with alpha-helices covering one face. The other side of the sheet is largely solvent-exposed and contains the active site. The C-terminal domain comprises a six-stranded, antiparallel beta-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 C. elegans unc-104 gene encodes a putative kinesin heavy chain-like protein.

Mutations in the unc-104 gene of the nematode C. elegans result in uncoordinated and slow movement. Transposon insertions in three unc-104 alleles (e2184, rh1016, and rh1017) were used as physical markers to clone the unc-104 gene. DNA sequence analysis of unc-104 cDNAs revealed an open reading frame capable of encoding a 1584 amino acid protein with similarities to kinesin heavy chain. The similarities are greatest in the amino-terminal ATPase and microtubule-binding domains. Although the primary sequence relatedness to kinesin is weak in the remainder of the molecule, the predicted secondary structure and regional isoelectric points are similar to kinesin heavy chain.

Crystallization of the bifunctional biotin operon repressor.

The bifunctional birA gene product, BirA, which represses the biotin biosynthetic bio operon and also activates biotin in Escherichia coli, has been crystallized. The crystals have the tetragonal space group P4(1)2(1)2, or its enantiomorph, with unit cell dimensions a = b = 114.0 A and c = 60.2 A and diffract to at least 2.3 A resolution. The crystal packing requires that the monomers of the birA protein be arranged as dimers with two-fold symmetry. BirA is the first protein to be crystallized that is both a transcriptional regulator and an enzyme.

The nucleotide sequence of the Escherichia coli rts gene.

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.

The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon.

The nucleotide sequence of the biotin (bio) biosynthetic operon of Escherichia coli has been determined. The 5.8-kilobase region contains the five biotin operon genes, bioA, B, F, C, and D. and an open reading frame of unknown function. The operon is negatively regulated and divergently transcribed from a control region between the bioA and bioB genes. The product of the bioA gene, 7,8-diaminopelargonic acid aminotransferase, was discovered to be related to ornithine aminotransferase. The product of the bioF gene, 7-keto-8-aminopelargonic acid synthetase, was found to be similar to 5-aminolevulinic acid synthetase.

Overproduction and rapid purification of the biotin operon repressor from Escherichia coli.

The Escherichia coli biotin operon repressor protein (BirA) has been overexpressed at the level of 0.5-1% of the total cellular protein from the plasmid pMBR10. Four lines of evidence demonstrated that authentic BirA protein was produced. First, birA plasmids complemented birA mutants for both the repressor and biotin holoenzyme synthetase activities of BirA. Second, biotin holoenzyme synthase activity was increased in strains containing the overproducing plasmids. Third, deletion of sequences flanking the birA gene did not alter production of the 35-kDa BirA protein, but insertion of oligonucleotide linkers within the birA coding region abolished it. Fourth, the 35-kDa protein copurified with the biotin binding activity normally associated with BirA. The birA protein has been purified to homogeneity in a three-step process involving chromatography on phosphocellulose and hydroxyapatite columns.

Isolation and characterization of Escherichia coli birA intragenic suppressors.

The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions. Twenty-five heat-resistant revertants of three temperature-sensitive birA alleles (birA85, birA104 and birA879) were isolated and categorized into five growth and six repression classes. The revertants appear to increase biotin activation by raising the specific activity of BirA and/or increasing the number of enzyme molecules. The 19 birA85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations. The birA85 revertants included a novel class of bio superrepressor mutations. Repressor titration experiments suggested that many of the birA85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids. The majority of the birA104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the birA104 mutation has been substituted by the wild-type or a chemically similar amino acid.

DNA-binding and enzymatic domains of the bifunctional biotin operon repressor (BirA) of Escherichia coli.

The negative regulation of the biotin biosynthetic (bio) operon in Escherichia coli is mediated by the bifunctional birA gene product, which serves as the bio repressor and biotin-activating enzyme. Nucleotide sequence analysis of 18 mutations in the birA gene was employed to study the DNA-binding and enzymatic functions of the BirA protein. The results indicate that a predicted helix-turn-helix structure, from amino acid (aa) positions 18 to 39 within the 321-aa BirA protein, may be responsible for sequence-specific DNA binding, whereas the temperature-sensitive mutations affecting biotin activation are found in two regions from aa positions 83-119 and 189-235.

Nucleotide sequence of the birA gene encoding the biotin operon repressor and biotin holoenzyme synthetase functions of Escherichia coli.

A 2.2-kb region of DNA containing the birA gene of Escherichia coli has been sequenced. The birA gene sequence predicts a 35.3-kDal [321 amino acids (aa)] bifunctional protein containing biotin-operon-repressor and biotin-holoenzyme-synthetase activities. Mutations, generated by random insertion of XhoI linkers, defined the extent of the gene. Mutations affecting one or more of five discernable properties of birA [Barker, D. and Campbell, A., J. Bacteriol., 143 (1980) 789-800] were mapped. Three mutations that result in temperature-sensitive (ts) growth, birA85, birA215, and birA879 mapped in the N-terminal two-thirds of the protein. The birA352 mutation, which partially complements birA215 and birA879, maps in the N-terminal third of the protein. Finally, birA361 maps closest to the amino terminus.