Autoregulated instability of beta-tubulin mRNAs by recognition of the nascent amino terminus of beta-tubulin.

Tubulin synthesis in animal cells is controlled by an autoregulatory mechanism that modulates the stability of polysome-bound tubulin messenger RNAs. The beta-tubulin RNAs are selectively targeted as substrates for destabilization not through the recognition of specific RNA sequences, but rather through co-translational recognition of the amino-terminal beta-tubulin tetrapeptide after its emergence from the ribosome. This motif is likely to be used in other systems where RNA degradation is coupled to ribosome attachment and translation.

cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment.

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes.

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting.

Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen.

We have isolated a series of overlapping cDNA clones for approximately 95% of the mRNA that encodes CENP-B, the 80-kD human centromere autoantigen recognized by patients with anticentromere antibodies. The cloned sequences encode a polypeptide with an apparent molecular mass appropriate for CENP-B. This polypeptide and CENP-B share three non-overlapping epitopes. The first two are defined by monoclonal antibodies elicited by injection of cloned fusion protein. Epitope 1 corresponds to a major antigenic site recognized by the anticentromere autoantibody used to obtain the original clone. Epitope 2 is a novel one not recognized by the autoantibody. These epitopes were shown to be distinct both by competitive binding experiments and by their presence or absence on different subcloned portions of the fusion protein. The third independent epitope, recognized by a subset of anticentromere-positive patient sera, maps to a region substantially closer to the amino terminus of the fusion protein. DNA and RNA blot analyses indicate that CENP-B is unrelated to CENP-C, a 140-kD centromere antigen also recognized by these antisera. CENP-B is the product of a 2.9-kb mRNA that is encoded by a single genetic locus. This mRNA is far too short to encode a polypeptide the size of CENP-C. The carboxy terminus of CENP-B contains two long domains comprised almost entirely of glutamic and aspartic acid residues. These domains may be responsible for anomalous migration of CENP-B on SDS-polyacrylamide gels, since the true molecular mass of CENP-B is approximately 65 kD, 15 kD less than the apparent molecular mass deduced from gel electrophoresis. Quite unexpectedly, immunofluorescence analysis using antibodies specific for CENP-B reveals that the levels of antigen vary widely between chromosomes.

Sequence and expression of the chicken beta 5- and beta 4-tubulin genes define a pair of divergent beta-tubulins with complementary patterns of expression.

We have determined the nucleotide sequence of the chicken beta 5 (c beta 5)-tubulin gene. The gene displayed the coding structure common to all previously studied vertebrate beta-tubulin genes and was divided into four exon sequences interrupted by three intervening sequences (located between codons 19 and 20, within codon 56, and within codon 93). Comparison of the predicted polypeptide sequence encoded by c beta 5 with those of four other available chicken beta-tubulin sequences revealed that c beta 5 encoded a highly divergent beta-tubulin polypeptide isotype which was distinguished from previously known sequences primarily by two discrete variable sequence domains. However, c beta 5 uniquely shared identity in 16 residue positions with another divergent chicken beta-tubulin gene, c beta 4. These common sequences distinguished c beta 4 and c beta 5 from the remaining three chicken beta-tubulin genes. Analysis of the expression of c beta 5 and c beta 4 revealed a strikingly complementary pattern of gene expression: c beta 5 was expressed in a wide variety of cell and tissue types but not in neurons, whereas c beta 4 expression was detected uniquely in neuronal cells. Overall, these findings suggest the existence of two divergent families of beta-tubulin sequences in the chicken and further raise the possibility that the complementary expression of the c beta 4 and c beta 5 genes may fulfill a requirement for the presence of a divergent beta-tubulin polypeptide isotype in all cell types.

Sequence and expression of the chicken beta 3 tubulin gene. A vertebrate testis beta-tubulin isotype.

We report the determination of the complete DNA sequence for c beta 3, a chicken beta-tubulin gene which we show to be the dominant beta-tubulin expressed in testis. Like all previously studied vertebrate beta-tubulin genes, the gene is divided into four exon sequences interrupted by three intervening sequences (located between amino acids 19 and 20, within codon 56, and within codon 93). Analysis of the program of expression of this gene indicates that it encodes the dominant chicken testis beta-tubulin, although it is also expressed at lower levels in a wide variety of cell and tissue types. Comparison of the predicted polypeptide sequence for c beta 3 with four other available chicken beta-tubulin genes confirms our earlier suggestion that within an otherwise conserved framework, sequences within two variable region domains serve to define specific beta-tubulin polypeptide isotypes. The data indicate that the c beta 3 gene encodes a unique beta-tubulin isotype which diverges from the dominant neuronal beta-tubulin isotype in 18 of 445 residues (4%). Although the protein coding regions of the c beta 3 gene are highly homologous to the chicken c beta 1, c beta 2, c beta 4, and c beta 5 genes previously reported by us, no significant sequence homology with these previously analyzed genes is discernible in the 5'- or 3'-untranslated region sequences, in the intervening sequences, or in the presumptive transcriptional promoter sequences.