Isolation and characterization of human cDNA clones encoding the alpha and the alpha' subunits of casein kinase II

Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.     

Molecular cloning of the human casein kinase II alpha subunit

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II alpha subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the alpha subunits.     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

cDNA cloning, genomic structure, chromosomal mapping, and expression analysis of parotid secretory protein in pig

A novel cDNA has been isolated from pig parotid glands by 3' and 5' rapid amplification of cDNA ends and designated parotid secretory protein (PSP). The open reading frame of this cDNA covers 714 bases, encoding 238 amino acids, which show 56% identity with human PSP at the level of the primary protein structure. The PSP genomic sequence comprises eight exons and seven introns, is approximately 22 kb in size, determined by sequencing, and maps to pig chromosome 17q21-q23. RT-PCR, dot blot, and Northern blot analyses demonstrated that PSP is strongly expressed in parotid glands, but is not present in heart, liver, lung, kidney, muscle, or stomach. A search for functionally significant protein motifs revealed consensus sequences for casein kinase II phosphorylation and N-myristoylation. We observed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu-X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is similar to the leucine zipper.     

C-ski cDNAs are encoded by eight exons, six of which are closely linked within the chicken genome.

The c-ski locus extends a minimum of 65 kb in the chicken genome and is expressed as multiple mRNAs resulting from alternative exon usage. Four exons comprising approximately 1.5 kb of cDNA sequence have been mapped within the chicken c-ski locus. However, c-ski cDNAs include almost 3 kb of sequence for which the exon structure was not defined. From our studies using the polymerase chain reaction and templates of RNA and genomic DNA, it is clear that c-ski cDNAs are encoded by a minimum of eight exons. A long 3' untranslated region is contiguous in the genome with the distal portion of the ski open reading frame such that exon 8 is composed of both coding and noncoding sequences. Exons 2 and 3 are separated by more than 25 kb of genomic sequence. In contrast, exons 3 through 8, representing more than half the length of c-ski cDNA sequences, are closely linked within 10 kb in the chicken genome.     

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.     

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

Isolation and characterization of a cDNA and a pseudogene for mouse lactate dehydrogenase-A isozyme

A mouse lactate dehydrogenase-A cDNA was isolated and it was shown to contain the 393bp of the protein-coding sequence and 488bp of the 3' untranslated region. The amino acid sequence deduced from its open reading frame provided independent evidence for the sequence of residues 201-331 of mouse LDH-A subunit (muscle). This cDNA clone was used as a probe to isolate a mouse genomic clone containing a truncated, processed LDH-A pseudogene. This pseudogene showed 81.6% homology at 713 positions compared with the LDH-A cDNA sequence. The divergence of this pseudogene was estimated to have occurred 39 million years ago.     

Sequencing and exon mapping of the inositol 1,4,5-trisphosphate receptor cDNA from Drosophila embryos suggests the presence of differentially regulated forms of RNA and protein.

A single gene appears to code for the inositol 1,4,5-trisphosphate receptor (itpr) in Drosophila melanogaster, as compared to three known genes in mammals. Expression of the itpr gene in Drosophila occurs in a wide range of tissues and developmental stages, suggesting its requirement during diverse cellular and physiological processes. A head cDNA for the Drosophila IP3R has previously been cloned and sequenced. Here we present and analyse the sequence of cDNAs encoding the complete IP3R, obtained from embryonic stages. The embryonic cDNA is 10525bp long and is a splice variant of the head cDNA. It differs from the latter in three main respects. It has longer 5' and 3' untranslated regions, two potential casein kinase II sites are missing in the embryo form and it contains an alternate exon which results in the replacement of three residues (VHF) in the head form by five residues (GVGHSV) in the embryo form. The significance of these changes is discussed. An exon-intron map of the gene derived from sequencing of intron-containing genomic fragments is also presented. The gene has a total of 11 introns, of which more than half are clustered in a region of the modulatory domain of the IP3R.