A regulatory element upstream of the human myoglobin gene functions as a muscle-specific enhancer (MSE) in conjunction with core promoter elements of the myoglobin gene, but not in combination with the simian virus 40 (SV40) early promoter. These two promoters differ in the sequences of their 'TATA boxes': for the myoglobin gene, the sequence is TATAAAA, whereas for SV40, the sequence is TATTTAT. We have now tested the hypothesis that this sequence difference is responsible for the differential response of the promoters to the MSE. We found that when the TATA box sequence of the myoglobin promoter was changed to that of the SV40 promoter, responsiveness to the MSE was abolished; conversely, when the SV40 TATA box sequence was changed to that of the myoglobin promoter, the promoter became responsive to the MSE. We conclude that mammalian TATA-box elements are functionally heterogeneous, and suggest that this heterogeneity reflects differential interactions with distinctive TATA box-binding factors, only some of which can act cooperatively with MSE-binding proteins to generate an active transcriptional complex.

A chicken 3-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 (8-tubulin-specific regions and the direction of transcription were determined within each cloned fragment. One clone (503) contained a (-tubulin-specific region of6.8 kilobase pairs (kbp) that included three intervening sequences as well as a number of inverted repeat structures. The remaining clones contained (3-tubulin-specific sequences that were close to or, in two cases, substantially less than 1.9 kbp long. Because mature human .0-tubulin mRNA is approximately 1.9 kbp long, these short DNA regions cannot on their own encode a functional (3-tubulin mRNA. Analysis using 3'-and 5'-specific probes derived from the chicken cDNA clone showed the presence ofboth ofthese end regions within one truncated tubulinlike 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 f-tubulin mRNA.Microtubules are constituent parts of a diverse variety of eukaryotic subcellular structures. An integral part of the mitotic apparatus, cilia, flagella, and elements ofthe cytoskeleton, they consist principally of two soluble proteins, named a-and ,B-tubulin, each with a molecular weight ofabout 55,000. Work performed in this laboratory (1) has demonstrated that, in chickens, separate mRNAs encode a-and ,B-tubulin; thus,-these proteins are transcribed from distinct genes. Construction of cDNA clones from isolated chicken a-and /&tubulin mRNAs and use of these probes in Southern blotting experiments of whole genomic DNAs (2) suggested the presence of about 4 copies each per genome of a-and /3-tubulin genes in the chicken; the corresponding value in experiments using human DNA is about 14 copies per genome.The expression of a number of tubulin genes might account for the reported appearance of multiple a-and ,B-tubulins isolated from various sources (3-6). Alternatively, such heterogeneity may reflect posttranslational modifications of a protein transcribed from one or a few genes. The extent to which protein heterogeneity is reflected at the genetic level can only be adequately revealed by a direct examination of the structure and organization ofthe genes themselves. In addition, such an analysis is a necessary prerequisite to addressing questions relating to tubulin gene expression. Several lines of evidence-including protein sequence data (7), blot hybridization experiments (2), and the ability of a-and /-tubulins from different species to copolymerize (8)-strongly suggest that tubulins are highly evolutionarily conserved. This paper describes the isolation and characterization of cloned DNA fragments containing 3tubulin-specific sequences from...

The ability of a chicken a-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, 2a, contains an a-tubulin-specific region of 5.0 kilobases that includes three intervening sequences. The second clone, 19a, contains an a-tubulin-specific region of 5.4 kilobases and has somewhat diverged 5' and 3' ends. Clone 19a has only two intervening sequences that correspond to the first two in clone 2a. However, these intervening sequences differ in size between clones 2a and 19u and show no detectable sequence homology. The sumof the lengths of sequences in either clone that hybridize to the cDNA probe accounts for essentially the entire length ofthe cDNA molecule.A remarkable feature ofmicrotubules is the diversity of eukaryotic cellular functions with which they are associated. Included in such functions are mitosis, cell motility, intracellular transport, and secretion. The principal components of microtubules are two soluble proteins named a-and f-tubulin, each with a molecular mass of -55,000 daltons and each encoded by a separate gene or genes (1). Several observations suggest that the sequence oftubulin proteins is highly evolutionarily conserved. Hybrid microtubule structures may be formed by in vitro copolymerization of a-and (&tubulins from different species (2), NH2-terminal sequence data indicate considerable homology between sea urchin and chicken tubulins (3), and cDNA probes constructed from chicken a-and (B-tubulin mRNAs are able to cross-hybridize with genomic DNAs from a variety ofeukaryotic species under stringent conditions (4).How many genes encode a-and ,B tubulins, and what factors govern their expression? One approach to this question involves a direct examination of the genes themselves. The existence of multiple tubulins is implied by several reports of electrophoretic microheterogeneity in a-and /-tubulin proteins (5-8) and has been recently confirmed by protein sequence data that show multiple closely related forms of porcine a-tubulin (9). To isolate genomic sequences containing human a-tubulin genes, we have exploited the ability of a chicken a-tubulin cDNA probe to cross-hybridize with corresponding sequences from other eukaryotic species (4). We chose to study human tubulin gene organization because ofthe possibility that defective gene structure or expression might underlie certain disease conditions (10). We previously reported the isolation and characterization of several humanB-tubulin genes (11). This paper reports the structure of two human a-tubulin genes. MATERIALS AND METHODSScreening of Libraries, Restriction Mapping, and Southern Blot Analysis. The partial EcoRI (12) and par...

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