Isolation and partial sequencing of the human prothymosin alpha gene family. Evidence against export of the gene products

Prothymosin alpha and thymosin alpha 1 are believed to be thymus-derived, hormone-like materials with immunomodulatory functions performed outside the cell. These functions are inconsistent with the existence of a full length cDNA clone that does not encode an amino-terminal signal peptide or several consecutive hydrophobic residues. A study of the prothymosin alpha mRNAs and genes was undertaken in search of evidence for secreted forms of the protein. Prothymosin alpha mRNA was localized exclusively on free, rather than membrane-bound, polysomes. Upon screening cosmid and plasmid libraries totaling 2 X 10(6) clones, a gene family consisting of six members was identified. Sequence information from the 5'-ends of all the genes indicated that none encodes an amino-terminal signal peptide. One of the genes, apparently by means of alternate splicing, gives rise to two prothymosin alpha mRNAs, one of which has an additional internal glutamic acid codon with respect to the other. Comparison of the translated nucleic acid sequences of the five remaining genes with those encoded in the mRNAs revealed 30-98% homology in the first 50 amino acids. These five genes appear to be processed genes and/or pseudogenes. The localization of prothymosin alpha mRNAs on free polysomes, together with the partial nucleotide sequences of the genes, strongly suggest an intracellular function for prothymosin alpha. Therefore, the possibility must be raised that prothymosin alpha and its peptide derivatives act as xenobiotics when introduced into assays of immune function.     

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.     

The amino acid sequence of bovine thymus prothymosin alpha

Prothymosin alpha has been purified from calf thymus and its amino acid sequence determined. It contains 109 amino acid residues and closely resembles human prothymosin alpha, with only two substitutions, glutamic acid for aspartic acid at position 31 and alanine for serine at position 83. This is in contrast to six differences between rat and bovine prothymosins, including four substitutions and two deletions. The structural similarity of the bovine and human polypeptides makes the former a good candidate for studies on the evaluation of the biological activities of prothymosin alpha in human systems.     

Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.     

Human U1 small nuclear RNA genes: extensive conservation of flanking sequences suggests cycles of gene amplification and transposition.

The DNA immediately flanking the 164-base-pair U1 RNA coding region is highly conserved among the approximately 30 human U1 genes. The U1 multigene family also contains many U1 pseudogenes (designated class I) with striking although imperfect flanking homology to the true U1 genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb) regions of the human genome spanning U1 homologies. Two clones contain one true U1 gene each, and the third bears two class I pseudogenes 9 kb apart in the opposite orientation. We show by genomic blotting and by direct DNA sequence determination that the conserved sequences surrounding U1 genes are much more extensive than previously estimated: nearly perfect sequence homology between many true U1 genes extends for at least 24 kb upstream and at least 20 kb downstream from the U1 coding region. In addition, the sequences of the two new pseudogenes provide evidence that class I U1 pseudogenes are more closely related to each other than to true genes. Finally, it is demonstrated elsewhere (Lindgren et al., Mol. Cell. Biol. 5:2190-2196, 1985) that both true U1 genes and class I U1 pseudogenes map to chromosome 1, but in separate clusters located far apart on opposite sides of the centromere. Taken together, these results suggest a model for the evolution of the U1 multigene family. We speculate that the contemporary family of true U1 genes was derived from a more ancient family of U1 genes (now class I U1 pseudogenes) by gene amplification and transposition. Gene amplification provides the simplest explanation for the clustering of both U1 genes and class I pseudogenes and for the conservation of at least 44 kb of DNA flanking the U1 coding region in a large fraction of the 30 true U1 genes.     

Human prothymosin alpha: purification of a highly acidic nuclear protein by means of a phenol extraction

Human prothymosin alpha, virtually alone among proteins, is recovered from the aqueous phase of phenol-extracted cell lysates prepared from human myeloma cells or COS cells that were transfected with the human prothymosin alpha gene. This observation forms the basis for purification of the protein to homogeneity in two steps--phenol extraction followed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels to remove residual contaminants consisting chiefly of carbohydrate and RNA.     

Human U1 small nuclear RNA pseudogenes do not map to the site of the U1 genes in 1p36 but are clustered in 1q12-q22.

Human U1 small nuclear RNA is encoded by approximately 30 gene copies. All of the U1 genes share several kilobases of essentially perfect flanking homology both upstream and downstream from the U1 coding region, but remarkably, for many U1 genes excellent flanking homology extends at least 24 kilobases upstream and 20 kilobases downstream. Class I U1 RNA pseudogenes are abundant in the human genome. These pseudogenes contain a complete but imperfect U1 coding region and possess extensive flanking homology to the true U1 genes. We mapped four class I pseudogenes by in situ hybridization to the long arm of chromosome 1, bands q12-q22, a region distinct from the site on the distal short arm of chromosome 1 to which the U1 genes have been previously mapped (Lund et al., Mol. Cell. Biol. 3:2211-2220, 1983; Naylor et al., Somat. Cell Mol. Genet. 10:307-313, 1984). We confirmed our in situ hybridization results by genomic blotting experiments with somatic cell hybrid lines with translocation products of human chromosome 1. These experiments provide further evidence that class I U1 pseudogenes and the true U1 genes are not interspersed. The results, along with those published elsewhere (Bernstein et al., Mol. Cell. Biol. 5:2159-2171, 1985), suggest that gene amplification may be responsible for the sequence homogeneity of the human U1 gene family.