An amino-terminal motif functions as a second nuclear export sequence in BRCA1

Mutations in the breast cancer susceptibility gene 1 (BRCA1) account for a substantial percentage of familial breast and ovarian cancers. Although BRCA1 is thought to function within the nucleus, it has also been located in the cytoplasm. In addition, BRCA1 accumulates in the nucleus of cells treated with leptomycin B, an inhibitor of chromosome region maintenance 1-mediated nuclear export, indicative of its active nuclear export via this pathway. The nuclear export signal in BRCA1 has been described as consisting of amino acid residues 81-99. However, a number of other tumor suppressors have multiple nuclear export sequences, and we sought to determine whether BRCA1 did also. Here, we report that BRCA1 contains a second nuclear export sequence that comprises amino acid residues 22-30. By use of the human immunodeficiency virus-1 Rev complementation assay, this sequence was shown to confer export capability to an export-defective Rev fusion protein. The level of export activity was comparable with that of residues 81-99 comprising the previously reported nuclear export sequence in BRCA1. Mutation of leucine 28 to an alanine reduced nuclear export by approximately 75%. In MCF-7 cells stably transfected with a BRCA1 cDNA containing mutations in this novel sequence or the previously reported export sequence, BRCA1 accumulated in the nucleus. These data imply that BRCA1 contains at least two leucine-dependent nuclear export sequences.     

In-frame deletions of BRCA1 may define critical functional domains

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.     

Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling

The eye lens contains a structural protein, alpha crystallin, composed of two homologous primary gene products alpha A2 and alpha B2. In certain rodents, still another alpha crystallin polypeptide, alpha AIns, occurs, which is identical to alpha A2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete alpha A crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the alpha A gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5' non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the alpha AIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3' non-coding region. The second exon is bordered by a 3' splice junction (A X G/G X C), which deviates from the consensus for donor splice sites (A X G/G X T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.     

Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin.

Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109.     

BRCA1蛋白的序列及空间结构的分析

查询人的BRCA1蛋白的氨基酸序列,利用生物信息学的方法进行相似性搜索,获得一系列BRCA1蛋白的氨基酸序列。选择了其中的11条序列,对BRCA1蛋白进行了多重序列分析和进化分析,对BRCA1蛋白的BRCT结构域进行三维同源模型的构建与比较分析。分析结果表明:BRCA1中某些特定部位的氨基酸序列高度保守;确定氨基酸的保守位点并联合进化分析可对基因错义突变的致病性做初步地猜测;相近物种来源的BRCA1具有较近的亲缘关系,而且具有极其相似的三维空间结构。这些为研究BRCA1蛋白的结构与功能关系提供指导意义。     

Sequence of the gene for ribosomal protein L23 from the archaebacterium Methanococcus vannielii

The N-terminal sequence of HPLC-purified protein L23 from the Methanococcus vannielii ribosome has been determined by automated liquid-phase Edman degradation. Using the N-terminal amino acid sequence, an oligonucleotide probe complementary to the 5'-end of the gene was synthesized. The 26-mer oligonucleotide, containing two inosines, was used for hybridization with digested M. vannielii chromosomal DNA. The hybridizing band from HpaII-digested genomic DNA was ligated into pUC18 to yield plasmid pMvaZ1 containing the entire gene of protein L23. The nucleotide sequence complemented the partial amino acid sequence, and the gene codes for a protein of 9824 Da. The amino acid sequence of protein L23 form M. vannielii was compared to that of ribosomal proteins from other archaebacteria as well as from eubacteria and eukaryotes. The number of identical amino acids is highest when the M. vannielii protein is compared to the homologous protein from yeast and lowest vs that from tobacco chloroplasts. Interestingly, the secondary structures of the proteins as predicted by computer programs are more conserved than the primary structures.     

Proteolytic processing of the alpha-chain of the lysosomal enzyme, beta-hexosaminidase, in normal human fibroblasts

The two subunits of beta-hexosaminidase undergo many post-translational modifications characteristic of lysosomal proteins, including limited proteolysis. To identify proteolytic cleavage sites in the alpha-chain, we have biosynthetically radiolabeled the transient forms, isolated these by immunoprecipitation, gel electrophoresis, and electroelution, and subjected them to automated Edman degradation. The position of the NH2-terminal amino acid was inferred from the elution cycle of the radioactive amino acid and the primary sequence encoded in the alpha-chain cDNA. The amino terminus of the precursor obtained by in vitro translation of SP6 alpha-chain mRNA in the presence of microsomes was leucine 23. The same amino terminus was found in precursor alpha-chain synthesized by normal human fibroblasts (IMR90) in a 1- or 3-h pulse or secreted by these cells in the presence of NH4Cl. The alpha-chain isolated after a 3-h pulse followed by a 5-h chase (intermediate form) included a mixture of molecular species of which the amino terminus was arginine 87 (most abundant), histidine 88, or leucine 90. After a 20-h chase (mature form) the latter species predominated. This mature form of the alpha-chain remained fully reactive with antibody raised against the carboxyl-terminal 15 amino acids, indicating little if any proteolysis at the carboxyl terminus. Thus synthesis and maturation of the alpha-chain of beta-hexosaminidase includes two major proteolytic cleavages: the first, between alanine 22 and leucine 23, removes the signal peptide to generate the precursor form, whereas the second occurs between the dibasic amino acids, lysine 86 and arginine 87. The second cleavage is followed by trimming of 3 additional amino acids to give the mature form of the alpha-chain.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

The primary structure of rat ribosomal protein S28.

The amino acid sequence of the rat 40S ribosomal subunit protein S28 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein S28 has 69 amino acids and has a molecular weight of 7,836. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the S28 gene. The mRNA for S28 is about 450 nucleotides in length. Rat S28 is homologous to Saccharomyces cerevisiae S33.     

Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links

Cells deficient in the Werner syndrome protein (WRN) or BRCA1 are hypersensitive to DNA interstrand cross-links (ICLs), whose repair requires nucleotide excision repair (NER) and homologous recombination (HR). However, the roles of WRN and BRCA1 in the repair of DNA ICLs are not understood and the molecular mechanisms of ICL repair at the processing stage have not yet been established. This study demonstrates that WRN helicase activity, but not exonuclease activity, is required to process DNA ICLs in cells and that WRN cooperates with BRCA1 in the cellular response to DNA ICLs. BRCA1 interacts directly with WRN and stimulates WRN helicase and exonuclease activities in vitro. The interaction between WRN and BRCA1 increases in cells treated with DNA cross-linking agents. WRN binding to BRCA1 was mapped to BRCA1 452–1079 amino acids. The BRCA1/BARD1 complex also associates with WRN in vivo and stimulates WRN helicase activity on forked and Holliday junction substrates. These findings suggest that WRN and BRCA1 act in a coordinated manner to facilitate repair of DNA ICLs.     

Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase

The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approximately 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC–RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.     

DNA cloning and amino acid sequence determination of a major constituent protein of mammalian nucleoli

Using a cDNA probe encoding the nucleolar protein N038 of Xenopus laevis, we have isolated clones that code for the corresponding mammalian protein from cDNA libraries of mouse embryonal carcinoma and fetal liver cells. The murine cDNA-derived amino acid sequence defines a polypeptide of 292 amino acids (including the initial methionine) of a total molecular weight of 32560 and identifies a single 1.5 kb mRNA on Northern blot hybridization. This polypeptide, which is highly homologous to the Xenopus protein N038, displays an organization in three major domains: (1) an aminoterminal portion of 119 amino acids, which shows a striking homology to nucleoplasmin of Xenopus; (2) a central portion of 68 amino acids that contains two extended acidic domains, a shorter of 13 residues and a longer of 29 residues, separated by an interval enriched in positively charged amino acids; (3) a carboxyterminal portion of 105 amino acids, which is almost identical to the reported partial amino acid sequence of human and rat nucleolar protein termed B23. The sequence comparisons show that the murine protein is the mammalian counterpart to the nucleolar protein N038 of Xenopus and is compatible with the idea that both proteins N038 represent the amphibian and murine equivalents to the human and rat nucleolar phosphoprotein B23. Special sequence features and predicted conformations of this protein are discussed in relation to the specific localization and the possible functions of this major nucleolar protein.     

Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1

In this paper, we report the identification and molecular characterization of a splice variant of human Mnk1 which has been named as Mnk1b. Human Mnk1b mRNA is homologous to human Mnk1 mRNA but lacking a region corresponding to exon 19, which causes a change in the reading frame generating a stop codon. The resulting protein lacks the last 89 amino acids at the C-terminal region that are replaced by 12 amino acids with an entirely new sequence. The C-terminal end in Mnk1 corresponds to the extracellular signal-regulated kinase (ERK1/2) binding site. Although Mnk1b lacks this domain and, consequently, is not phosphorylated by ERK1/2, it is able, however, to phosphorylate eIF4E in vitro and in vivo in a mitogen-activated protein kinases-independent manner. This result suggests that Mnk1b may play a key role in regulating protein translation in the absence of stimuli. Interestingly, a significant population of cells shows Mnk1b within the nucleus whereas Mnk1 is always detected in the cytoplasm. This fact may be explained because Mnk1b maintains the nuclear localization signal (NLS) but lacks the nuclear export sequence (NES).     

The primary structure of rat ribosomal protein L23.

The amino acid sequence of the rat 60S ribosomal subunit protein L23 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L23 has 140 amino acids and a molecular weight of 14,856. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the L23 gene. The mRNA for the protein is about 600 nucleotides in length. Rat L23 is homologous to Saccharomyces cerevisiae L17a and related to Escherichia coli L14 and other members of the prokaryotic L14 family.