Differential expression of two neural cell-specific beta-tubulin mRNAs during rat brain development.

We recently demonstrated that beta-tubulin mRNA expression is regulated during rat brain development. This is manifested by a dramatic decrease in both 1.8- and 2.9-kilobase (kb) mRNAs when extensive neurite elongation is occurring. Coincident with these decreases is the increased production of a 2.5-kb mRNA. (J.F. Bond and S.R. Farmer, Mol. Cell. Biol. 3:1333-1342, 1983). In the present study, we have isolated and characterized three different cDNAs corresponding to beta-tubulin mRNAs (R beta T.1, R beta T.2, and R beta T.3). Hybridization of 3' untranslated region subclones of R beta T.1 and R beta T.2 cDNAs to RNA from a variety of rat tissues and cells revealed that these two cDNAs are neural cell specific. R beta T.1 corresponds to an abundant 1.8-kb mRNA expressed only at early stages of rat brain development. R beta T.2 corresponds to the 2.5-kb mRNA expressed at later stages. These data strongly suggest that there is differential expression of the beta-tubulin multigene family during rat brain development.     

Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs

cDNA clones for a fifth polypeptide of rat brain calmodulin-dependent protein kinase II were isolated and sequenced. The cDNA sequence encoded a polypeptide, designated delta, consisting of 533 amino acid residues with a molecular weight of 60,080. Comparison of amino acid sequences of this and alpha, beta, beta', and gamma polypeptides of calmodulin-dependent protein kinase II reveals marked homology among them. The mRNAs for delta were expressed in rat brain tissues with different regional specificities. The distribution of alpha, beta/beta', gamma, and delta mRNAs in cerebrum, skeletal muscle, diaphragm, heart, small intestine, uterus, aorta, liver, kidney, lung, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.9-kilobase (kb) RNA species hybridizable with a probe for gamma was found in all the tissues examined, and 4.0-4.2-kb RNA species hybridizable with a probe for delta were found in all the tissues examined except for liver, while a 4.8-kb RNA species hybridizable with a probe for alpha and a 4.2-kb RNA species hybridizable with a probe for beta were present in brain but not in the other tissues. With the alpha probe, however, a 4.1- and 2.6-kb RNA species were both detected in skeletal muscle and diaphragm. With the beta probe, a 4.3-kb RNA in skeletal muscle and diaphragm, 2.9-kb RNA in small intestine, and 4.0-kb RNA in testis were detected. With the delta probe, a 3.5-kb RNA in heart and 1.8-kb RNA in testis were detected. Thus, gamma and delta mRNAs were expressed in various tissues, while alpha and beta/beta' mRNAs were primarily, if not exclusively, expressed in brain.     

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

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.     

Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes

In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.     

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.     

Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the previously determined sequences of beta-caseins from several other species. Approximately 38% of the amino acids have been conserved among the rat, ovine, bovine and human sequences and these conserved amino acids occurred in clusters throughout the protein. One such cluster containing the majority of the potential casein phosphorylation sites was located near the amino terminus. Contrary to the considerable divergence observed for the processed beta-casein, 14 of 15 amino acids in the signal peptide sequence of the precasein were identical between the rat and ovine caseins.     

Isolation and characterization of a cDNA clone encoding rat 5-lipoxygenase

A full-length cDNA clone encoding 5-lipoxygenase, a key enzyme in the formation of leukotrienes, was isolated from a rat basophilic leukemia cell lambda gt11 cDNA library. The 2.5-kilobase (kb) cDNA insert, whose identity was confirmed by hybrid-select translation and DNA sequence analysis, has a 2.0-kb open reading frame encoding a protein of Mr approximately 77,600 and includes 60 base pairs of 5'-untranslated region and 0.4 kb of 3'-untranslated region to the polyadenylation signal. The deduced amino acid sequence shows significant homology with published sequences for the rabbit reticulocyte lipoxygenase and soybean lipoxygenase-1; it also contains sequences similar to a consensus sequence found in several calcium-dependent membrane-binding proteins. The cDNA recognizes a 2.6-kb mRNA species which is detected in all tissues but is particularly abundant in RNA from lung.     

Sequence of an expressed human beta-tubulin gene containing ten Alu family members

The complete sequence of a functionally expressed human beta-tubulin gene (5 beta) is presented. The amino acid sequence encoded by this gene constitutes a distinct isotype, differing from a previously described human beta-tubulin sequence at 21 positions throughout the polypeptide chain. The beta-tubulin coding sequence in 5 beta is interrupted by three intervening sequences of 1014, 117 and 4826 nucleotides. The largest of these contains ten members of the Alu family of middle repetitive sequences. Together, these regions account for sixty percent of this intervening sequence. Two of the Alu elements are juxtaposed head to tail, and share the same flanking direct repeat. The ten Alu sequences are substantially divergent, both from each other and from an Alu consensus sequence, and several contain deletions of up to half the entire sequence.