The sequence of a human P-tubulin cDNA clone (D,B-1) is described; our data revealed 95.6% homology compared with the sequence of a human f-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 1-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 1-tubulin sequence (5,B) possessed a very high degree of homology with chicken and pig 3-tubulins in this region. Thus, human cells appear to contain two distinct ,B-tubulin isotypes. Both the intact ,B-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 53 genomic sequence also detected a 2.6-kilobase 1-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 ,B-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 f-tubulin mRNAs.a-and P-tubulins are the major soluble proteins of microtubules. These long filamentous structures are found in all eucaryotic cells and are involved in a remarkable diversity of cellular functions (11). In humans, each of the genes encoding a-and ,-tubulins constitutes a large multigene family of about 15 to 20 members (8), the majority of which have been isolated in recombinant fragments (9, 41). Sequence analyses of several of these genes have shown that many of them are pseudogenes (that is, sequences which contain one or more genetic lesions that preclude the synthesis of a functional protein product). Among these pseudogenes is a novel class that is characterized by (i) the absence of intervening sequences, (ii) the presence of a short polyadenylic acid [poly(A)] tract 3' to the poly(A) signal, and (iii) the existence of short flanking direct repeat sequences (22,42,43). These pseudogenes most probably arose by reintegration into the host germ line of a cDNA transcript synthesized on a processed mRNA template.Because a significant number, perhaps the majority, of human a-and P-tubulin genes are pseudogenes, an important question concerns the number offunctionally expressed sequences.One approach to this problem is to investigate the complexity of tubulin mRNAs by constructing cDNA clones. In this paper we describe the sequence of a human P-tubulin cDNA clone that encompasses 98.4% of the coding region. Our data revealed very extensive homology with the sequence of a processed human P-tubulin pseudogene and enabled us to estimate the time of insertion of the mRNA-derived molecule into the germ li...

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Background The pituitary gland is a neuroendocrine organ containing diverse cell types specialized in secreting hormones that regulate physiology. Pituitary thyrotropes produce thyroid-stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate, but the transcriptomic and epigenomic landscapes of these neuroendocrine cells have not been characterized. The goal of this work was to discover transcriptional regulatory elements that drive thyrotrope fate. Results We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an undifferentiated Pou1f1 lineage progenitor (GHF-T1) and a committed thyrotrope line that produces TSH (TαT1). We compared RNA-seq, ATAC-seq, histone modification (H3K27Ac, H3K4Me1, and H3K27Me3), and POU1F1 binding in these cell lines. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding at unique sites. We validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element. Conclusion These results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis. Graphical abstract

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.