Faculty Bibliography

To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement

A 3' untranslated region subclone from a human beta-tubulin cDNA clone has been used to dissect the human beta-tubulin multigene family. Four different beta-tubulin sequences were obtained. One consists of an expressed gene that yields two mRNA species of 1.8 kb and 2.6 kb as a consequence of alternative polyadenylation sites. The three remaining beta-tubulin sequences are all intronless pseudogenes, each containing a 3' poly(A) tract downstream from the poly(A) signal, and each flanked by a different short direct repeat. Two of these sequences were derived by integration into the host germ line of cDNA copies of the 1.8 kb mRNA; the third was derived from the 2.6 kb mRNA. Comparison of the functional and nonfunctional sequences suggests that the integration events took place 4, 10, and 13 million years ago. We anticipate that, in multigene families where germ-line expression occurs, a significant portion of sequences will be accounted for by pseudogenes generated via an RNA intermediate

The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs

The sequence of two human beta-tubulin pseudogenes is described. One contains an intervening sequence but lacks sequences encoding the 55 N-terminal amino acids of the polypeptide chain. A second has no introns but has a polyadenylate signal and an oligoadenylate tract at its 3' end, and it is flanked by a short direct repeat. These sequences have arisen by different mechanisms, including one that probably involves reverse transcription of a processed messenger RNA and reintegration of the complementary DNA copy into the genome

The ability of a chicken alpha-tubulin cDNA probe to cross-hybridize with human DNA under stringent conditions has been exploited to screen two independently constructed human genomic libraries. Nine clones were isolated, accounting for 60% of the bands observed in a whole genomic Southern blot of human DNA. Two clones were selected for further analysis by restriction mapping, orientation experiments using 3'- or 5'-specific probes, and electron microscopy of heteroduplexes. One clone, 2 alpha, contains an alpha-tubulin-specific region of 5.0 kilobases that includes three intervening sequences. The second clone, 19 alpha, contains an alpha-tubulin-specific region of 5.4 kilobases and has somewhat diverged 5' and 3' ends. Clone 19 alpha has only two intervening sequences that correspond to the first two in clone 2 alpha. However, these intervening sequences differ in size between clones 2 alpha and 19 alpha and show no detectable sequence homology. The sum of the lengths of sequences in either clone that hybridize to the cDNA probe accounts for essentially the entire length of the cDNA molecule

A chicken beta-tubulin cDNA probe has been used to screen two independently generated human genomic libraries. Of 13 EcoRI fragments detectable in a human genomic Southern blot experiment, 7 correspond in size to EcoRI fragments isolated from recombinant bacteriophage. The location of beta-tubulin-specific regions and the direction of transcription were determined within each cloned fragment. One clone (5 beta) contained a beta-tubulin-specific region of 6.8 kilobase pairs (kbp) that included three intervening sequences as well as a number of inverted repeat structures. The remaining clones contained beta-tubulin-specific sequences that were close to or, in two cases, substantially less than 1.9 kbp long. Because mature human beta-tubulin mRNA is approximately 1.9 kbp long, these short DNA regions cannot on their own encode a functional beta-tubulin mRNA. Analysis using 3'- and 5'-specific probes derived from the chicken cDNA clone showed the presence of both of these end regions within one truncated tubulin-like sequence. A second short tubulin-specific region failed to hybridize with a 3'-specific probe. These short sequences are therefore likely to be examples of pseudogenes that have arisen by loss of a portion of DNA essential to the production of functional human beta-tubulin mRNA