The First Intron of Human c-fms Proto-oncogene Contains a Processed Pseudogene (RPL7P) for Ribosomal Protein L7

During sequence analysis of the first intron of the human c-fms oncogene, we identified an open reading frame encoding the ribosomal protein L7 (RPL7). The presence of this sequence within intron 1 of the c-_fms gene was confirmed by Southern blot hybridization and by sequence analysis of two independent cosmid clones (cos2-e and cos1-22) that span the human genomic c-_fms locus. The RPL7 sequence was detected in a region of sequence overlapped by the cos2-e and cos1-22 cosmid clones but oriented opposite to the c-fms gene. We demonstrated that the sequence is identical to the full-length RPL7 cDNA sequence, but lacks any recognizable introns, has a 30-bp poly(A) tail, and is bracketed by two perfect direct repeats of 14 bp. We also showed that despite the fact that the 5′ flanking region of the RPL7 sequence contains a potential TATA box upstream of an intact open reading frame, this pseudogene (RPL7P) is not actively transcribed.     

Cloning and sequence analysis of an Ig lambda light chain mRNA expressed in the Burkitt''s lymphoma cell line EB4.

A cDNA library was constructed from the mRNA of the Ig lambda producing Burkitt's lymphoma cell line, EB4. Overlapping clones encompassing the coding sequence of the Ig lambda mRNA were isolated and sequenced. The predicted amino acid sequence shows a short hydrophobic leader peptide and a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The V region belongs to subgroup VI and has greatest homology (80%) with the Amyloid-AR protein. The constant region is the Kern- Oz+ isotype. Probing normal human DNA with the subcloned V lambda coding sequence detects one gene at high stringency and a family of 11 members at low stringency. To date, no restriction enzyme site polymorphisms have been detected. The V lambda VI gene is rearranged on both chromosomes of EB4 and is deleted on both chromosomes in the Burkitt's lymphoma cell line BL2.     

The Precursor of Differentiation Factor HLDF and Ribosomal Protein RPS21 Have a Common N-Terminal Sequence

The mature differentiation factor HLDF, isolated from cultural medium, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48–50 and 100–102 (numbering according to the structure of S21), i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21.     

The primary structure of rat ribosomal protein S5. A ribosomal protein present in the rat genome in a single copy.

The amino acid sequence of the rat 40 S ribosomal subunit protein S5 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by the determination, directly from the protein, of 17 residues near the NH2 terminus. S5 has 204 amino acids; the molecular weight is 22,863. The protein designated S5a has the same amino acid sequence as S5 except that it lacks the NH2-terminal 5 residues. It is not known whether the conversion of a portion of S5 to S5a is physiological or fortuitous. The mRNA for S5 has about 820 nucleotides. Hybridization of the S5 cDNA to digests of nuclear DNA indicates that the rat genome has only a single copy of the gene; this is in distinction to the mouse and human genomes which have three to six copies of the S5 gene. Rat ribosomal protein S5 is related to the eubacteria, the arachaebacteria, and the chloroplast family of S7 ribosomal proteins. There is a peptide of 16 residues at the carboxyl terminus of S5 that is highly conserved in 18 species spanning the three kingdoms and chloroplasts.     

Precursors of HLDF differentiation factor and ribosomal protein RPS21 have a common N-terminal sequence

The mature differentiation factor HLDF, isolated from culture fluid, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48-50 and 100-102, i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and the S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.     

A processed pseudogene with an intact coding sequence for rat liver cytochrome c oxidase subunit VIc

The biogenesis of eukaryotic cytochrome c oxidase involves the coordinate expression of nuclear and mitochondrial genes. Very little information is available on the gene structure of nuclear-coded cytochrome c oxidase subunits in mammalian systems. We report here the isolation and complete nucleotide sequence determination of a processed pseudogene for cytochrome c oxidase subunit VIc from rat liver. The pseudogene lacks introns and the coding region is intact with no deleterious lesions; however, there are 7 amino acid (aa) differences when compared to the sequence derived from cDNA clones. The pseudogene has the potential to code for a protein of 76 aa, containing a putative 3 aa N-terminal presequence when compared to the mature bovine heart VIc subunit. Potential regulatory regions, including a TATA box, are present in the 5'-flanking region.     

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS

Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.     

Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library

Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA.