Abstract: In the course of studying the natural immunity of cattle and other rural ruminants in Egypt, SOD1 was one of the genes under investigation. The nucleotide sequence analysis of the obtained SOD1 amplified segments of cattle lead to the identification and assignment of SOD1 retropseudogenes Ψ 71.4 and 69.1 on human chromosome 8 and 16, respectively. Sequencing and Blast analysis for SOD1 amplified segments (370 nt) of Egyptian native cattle cDNA and genomic DNA revealed that these segments have 97% similarity. Blast analysis showed that the amplified sequence of cattle genomic DNA has great similarity with Bos taurus SOD-1 mRNA; with four separate segments of Bos taurus chromosome 1 genomic superoxide dismutase 1 and showed high identities with DNA of Bos taurus chromosome 13 similar to SOD1. These results indicate that cattle genomic DNA has intronless pseudogene on Bos taurus chromosome 13. The amplified sequence of cattle genomic DNA also showed a high similarity with three separate segments of human chromosome 21 on which the SOD1 gene is known to be located. In addition, four interesting alignments were also observed; it showed high alignment with a segment of human chromosome 16; a segment of human chromosome 8 and with human SOD1 processed pseudogenes Ψ 69.1 and 71.4. The SOD1 processed pseudogene Ψ 69.1 can be assigned to human chromosome 16 and the SOD1 processed pseudogene Ψ 71.4 can be assigned to chromosome 8. Two segments of 2000 nt from both human chromosome 16 and 8 were used for homology search. The maches obtained have high identities with the whole length of human SOD1 processed pseudogenes Ψ 69.1 and 71.4, respectively. These results identify the complete SOD-1 retropseudogenes sequence and their location on human chromosome 16 and 8, respectively. They bear the structural hallmarks of processed pseudogene; they lack introns; the 5’ and 3’ direct repeats are presented. They also contain TATA box upstream from the 5’ direct repeat in a position almost identical to the functional TATA box. They also contain a 3’ poly (A or T)-rich stretch and are flanked by short direct repeat sequences. The presence of SOD1 retropseudogenes on human chromosome 16 and 8 reported in this study explain the reported FALS and other diseases in individuals with a normal chromosome 21.
INTRODUCTION
There are several isoforms of Superoxide Dismutase (SOD) that differ in metal binding ability, distribution in different cell compartments and in sensitivity to various reagents. Among these, Cu/Zn Superoxide Dismutase (SOD1) is widely distributed and comprises 90% of the total SOD (Noor et al., 2002). Bovine Cu/Zn superoxide dismutase gene was assigned to bovine chromosome 1 (Schmutz et al., 1996), whereas, bovine Cu/Zn SOD1 pseudogene was assigned to bovine chromosome 13 (Gallagher et al., 1999). In human, Cu/Zn superoxide dismutase gene was assigned to chromosome 21q in the segment enclosing the distal part 21q21-21q22.1 (Huret et al., 1987).
Noor et al. (2002) described the role of SODs, especially SOD1, in several diseases such as Familial Amyotrophic Lateral Sclerosis (FALS); Parkinson’s disease; Alzheimer’s disease; dengue fever; cancer; Down’s syndrome; cataract and several neurological disorders. Mutations in the SOD1 gene have been found to lead to the development of FALS (Andersen et al., 2003; Hough et al., 2004).
However approximately 80% of FALS cases could not be related to SOD1 mutations; thus, other candidate genes have been tested for their possible role in disease pathogenesis. The other isoforms of SOD-that is, MnSOD (SOD2) and extracellular SOD (SOD3)-have been examined, but none could be linked to FALS (de Belleroche et al., 1995; Siddique and Deng, 1996).
The abundance of pseudogenes is a remarkable feature of mammalian genomes. Pseudogenes are copies of specific genes and are present in every mammalian chromosome (Gonçalves et al., 2000; Chen et al., 2002). In general, pseudogenes are thought to be nonfunctional (Mighell et al., 2000) as they have accumulated vast numbers of mutations during evolution and have lost the ability to be transcribed. Pseudogenes fall into two distinct categories depending on the mechanism by which they are generated: Processed pseudogenes are reverse transcribed from mRNAs (and thus do not contain introns) whereas nonprocessed pseudogenes arise from duplications of genomic DNA (Mighell et al., 2000; Harrison et al., 2002a). Among the abundant processed pseudogenes, there are a substantial number of processed gens or retrogenes of novel function that also derive from mRNAs of various intron-containing genes (Brosius, 1999; Lahn and Page, 1999; Betra`n et al., 2002). A number of retropseudogenes have also been implicated in various human diseases (Zhang et al., 2003). Parental genes of human processed pseudogenes are of various types, including those for enzymes, structural proteins and regulatory proteins such as ligand-binding proteins and transcription factors (Ohshima et al., 2003). It is unclear how many pseudogenes exist in the human genome. Estimates for the number of human genes range from ~22,000 to ~75,000 (Harrison et al., 2002b). It is thought that up to 22% of these gene predictions may be pseudogenic (Lander et al., 2001; Yeh et al., 2001).
It is important to characterize the human pseudogene population, as their existence interferes with gene identification and annotation. They are also an important resource for the study of the evolution of protein families. In the course of studying the natural immunity of cattle and other rural ruminants in Egypt, SOD1 was one of the genes under investigation. The nucleotide sequence analysis of the obtained SOD1 amplified segments of cattle lead to the identification and assignment of SOD1 retropseudogenes 71.4 and Ψ 69.1 on human chromosome 8 and 16.
MATERIALS AND METHODS
Collection of samples and genomic DNA extraction: Blood samples of Egyptian native cattle were collected in Ethylene Diamine Tetra Acetic Acid (EDTA) and were processed as soon as possible. Genomic DNA was extracted from leucocytes following the established protocols (Blin and Stafford, 1976) modified by Shih and Weinberg (1982). DNA was finally dissolved in an appropriate volume (200 μL) of 1x Tris-EDTA (TE). Using a spectrophotometer, the DNA concentration was determined and diluted in sterile water to a concentration of 50 ng μL-1.
Total RNA extraction and reverse transcription: Total RNA from blood was extracted using Trifast reagent after RBCs hydrolysis. To assess the quantity and purity of the RNA isolates, spectrophotometrically, readings of each total-RNA were taken at 260 and 280 nm. All RNA sample A°260/A°280 ratios fell between 1.75 and 1.9, indicating very pure RNA in all cases. The quality of each RNA sample has been assessed by denaturing agarose gel electrophoresis and ethidium bromide staining. Two sharp bands (28-S and 18-S ribosomal RNA bands) segregated at the proper size (Grubor et al., 2004). First Strand cDNA synthesis has been conducted using READY TO GO YOU prime-First Strand Beads kit (Amersham).
PCR reactions: A PCR cocktail consisting of 1.0 μM upper and lower primers and 0.2 mM dNTPs, 10 mM Tris (pH 9.0), 50 mM KCL, 1.5 mM MgCl2, 0.01% gelatin (W/V), 0.1 triton X-100 and 1.25 unit of taq polymerase was aliquoted into tubes with 100 ng DNA or cDNA. The samples were subjected to 35 cycles of PCR amplification. Each cycle includes denaturation at 95°C for 1 min, annealing at 58°C for 2 min and primer polymerization at 72°C for 1 min. The SOD1 primers were 5-GTG CTG AAG GGC GAC G-3 for sense and 5-TTT CCA CCT TTG CCC AAG-3 for antisense (Lyons et al., 1997).
PCR products were subjected to 1.5% agarose gel electrophoresis and stained with ethidium bromide. The gels were examined with a UV lamp at a wave length of 312 nm and were photographed using Mp4 plus Polaroid Camera.
The internal sequencing of SOD1 from both genomic DNA and cDNA has been performed using ABI PRISM version 3.7. Sequence alignment was carried out using NCBI-BlASTN 2.2.13 version (Altschul et al., 1997).
RESULTS
In the present investigation, a pair of primers specific for SOD1 used to test for the presence of this gene on total RNA and genomic DNA extracted from blood of native cattle in Egypt, using the PCR and Reverse Transcription (RT-PCR) techniques. A single sharp band at approximately 370 bp is obtained with both cDNA and genomic DNA (Fig. 1).
NCBI-Blast analysis of the internal sequence of SOD1 amplified PCR product
(amplicon) of genomic DNA (Fig. 2) and of SOD1 amplified cDNA
(Fig. 3) showed a 97% alignment (Fig. 4).
The internal sequence analysis of SOD1 amplicon obtained from the cattle genomic
DNA showed a 98% alignment to mRNA of Bos taurus SOD1 sequences of the Gene
Bank database (gene bank accession numbers, gi: 31341527, 7356542 and 162960)
Fig. 5, represents the alignment of DNA with mRNA of Bos taurus
SOD1 gi: 31341527 as an example). The obtained amplicon from the cattle genomic
DNA also shares sequence homology with mRNA from other species such as Homo
sapiens gi: 48762945 (87%), Capra hircus gi: 5865327 (95%) and Mucaca
mulatto gi: 74136166 (91%).
| Fig. 1: | Representive ethidium bromide-staind gel of amplified PCR products of SOD1 using cDNA and DNA extracted from blood Egyptian native cattle, L 100 bp and the left arrow indicate the amplified fragments size |
Figure 6 represents the alignment with Homo sapiens.
The amplicon sequence of the native cattle genomic DNA from nucleotide (nt) 15 to 306 aligns with four separate segments of Bos taurus chromosome 1 genomic contig ref |NW-929793.1| superoxide dismutase 1 (Fig. 7). Eighty one nucleotides (from 15 to 95 nt) showed 96% alignment with a segment of exon 4 of the Bos taurus chromosome 1 from nt coordinate 10406 to 10326 (Fig. 7A); 73 nucleotides (93 to 165) showed 98% sequence homology with exon 3 from nt coordinate 9501 to 9429 (Fig. 7B); 96 nucleotides (163 to 258) showed 98% sequence homology with a segment of exon 2 from nt coordinate 7649 to 7554 (Fig. 7C) and 53 nucleotides (254 to 306) showed 98% sequence homology with a segment of Bos taurus chromosome 1 from nt coordinate 3959 to 3908 (Fig. 7D). It also shares sequence homology (99%) with Bos taurus chromosome BTA13 genomic contig ref |NW-928647.1| similar to Superoxide dismutase (Fig. 8).
Moreover, blast analysis of the SOD1 amplicon sequence (370 nt) of the native cattle genomic DNA aligns with three separate segments of DNA on human chromosome 21, gi: 7768676 (Fig. 9) with sequence identities of 86, 94 and 83%. These three separate segments represent exon four (Fig. 9A); exon three (Fig. 9B) and exon two (Fig. 9C), respectively. In addition four interesting alignments are found; alignment of the amplicon of the cattle genomic DNA fragment showed 78% identities (Fig. 10) with human SOD1 processed pseudogene Ψ 69.1 (gi: 181206); 84% alignment (Fig. 11) with processed pseudogene Ψ 71.4 (gi: 180712); 80% alignment (Fig. 12) with human chromosome 16 DNA from nt coordinate 94952 to 95227 (gi: 29124036) and 84% alignment with chromosome 8 (gi: 21307426) from nt coordinate 70966 to 71135 (Fig. 13).
Study of the alignment of the two processed pseudogenes with human chromosomes 8 and 16 reveals that processed pseudogene Ψ 69.1 (gi: 181206) is 94% identical with chromosome 16 and only 80% with chromosome 8. Whereas the processed pseudogene Ψ 71.4 (gi: 180712) is 98% identical with chromosome 8 and 80% alignment with chromosome 16.
A series of alignment was carried out in order to identify the complete sequence of the SOD1 pseudogenes 69.1 and Ψ 71.4 on human chromosome 16 and 8, respectively. A segment of 2000 bases of chromosome 16 (from nt coordinate 94000 to 96000 which comprises the segment of human chromosome 16 (from nt coordinate 94952 to 95227) that gave an 80% alignment with the investigated amplicon is tested for alignment with the human SOD1 processed pseudogenes Ψ 69.1 (gi: 181206). Pair-wise blast analysis showed that a segment of 713 bases (from nt coordinate 94713 to 95423 including 2 gaps at 94803 and 95040) has 94% homology and a small fragment of 36 nt (from 94647 to 94682) showed 100% alignment. The 30 nt inbetween (from nt coordinate 94683 to 94712) failed to give significant alignment with Ψ 69.1 SOD1 processed pseudogene (Fig. 14). This segment found to be a poly A sequence (Fig. 16).
The whole sequence of retropseudogene on chromosome 16 has high identities with exon 2, 3, 4 (82, 86 and 82%, respectively) and partially with exon 5 (88%) and no alignment with exon 1 of SOD1 normal gene on human chromosome 21 (Fig. 15). The complete sequence (777 nt) of SOD1 retropseudogene is represented in Fig. 16.
Similarly, NCBI-Blast analysis of a 2000 bases (from 70000 to 72000) that comprises
the human segment of chromosome 8 (from 70966 to 71135) which gave 84% alignment
with the investigated amplicon is tested for alignment with processed pseudogene
Ψ 71.4 (gi: 180712). The alignment shows that a segment of 668 bases (from
nt sequence 70671 to 71338) has 98% alignment and a small fragment of 31 nt
(from nt coordinate 70626 to 70656) has 100% identity (Fig. 17).
| Fig. 2: | Nucleotide sequences of the reverse strand superoxide dismutase-1 (SOD1) for the amplified PCR product of DNA extracted from blood Egyptian native cattle |
| Fig. 3: | Nucleotide sequences of the reverse strand superoxide dismutase-1 (SOD1) for the amplified RT-PCR product of mRNA extracted from blood Egyptian native cattle |
| Fig. 4: | Alignment of the SOD1 amplified fragment
of cDNA (a) with the SOD1 amplified fragment of DNA (b) extracted from
Egyptian native cattle, Identities = 271/277 (97%) Strand = Plus/Plus |
| Fig. 5: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA (a), with Bos taurus superoxide dismutase 1, soluble (SOD1), mRNA (b) gi: 31341527, Identities = 283/288 (98%), Gaps = 1/288 (0%) Strand = Plus/Minus |
| Fig. 6: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA (a) with Homo sapiens (b) superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult) (SOD1), mRNA gi: 48762945, Identities = 187/214 (87%), Gaps = 0/214 (0%) Strand = Plus/Minus |
| Fig. 7: | Alignment of the SOD1 amplified fragment of DNA cattle with Bos taurus chromosome 1 genomic contigref|NW_929793.1|Bt1_WGA5_2 superoxide dismutase 1, soluble Length = 691109 |
| Fig. 8: | Alignment of the SOD1 amplified fragment of DNA cattle (a) with Bos taurus chromosome 13 (b) genomic contig ref |NW_928647.1|Bt13_WGA2317_2 similar to Superoxide dismutase Identities = 291/292 (99%), Gaps = 1/292 (0%) |
| Fig. 9: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA with human chromosome 21 gi: 7768676, Strand = Plus/Minus |
| Fig. 10: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA with Human Cu/Zn processed pseudogene Y 69.1 gi: 181206, Identities = 169/214 (78%), Gaps = 7/214 (3%). Strand = Plus/Minus |
| Fig. 11: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA with Human Cu/Zn processed pseudogene y 71.4 gi:180712, Identities = 149/177 (84%), Gaps = 3/177 (1%) Strand = Plus/Minus |
| Fig. 12: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA with human chromosome 16 gi: 29124036, Identities = 223/278(80%), Gaps = 5 /278 (1%). Strand = Plus/Plus |
| Fig. 13: | Alignment of the SOD1 amplified fragment of Egyptian native cattle DNA with Human chromosome 8 gi:21307426 Identities = 146/172 (84%), Gaps = 2/172 (1%) Strand = Plus/Plus |
| Fig. 14: | Alignment of human chromosome 16 gi: 29124036 from nt coordinate 94000 to 96000 with Human Cu/Zn processed pseudogene y 69.1 gi: 181206 |
| Fig. 15: | Alignment of SOD1 retropseudogene on human chromosome 16 with complete sequence of human chromosome 21 gi: 7768676 |
| Fig. 16: | The full sequence of SOD1 retropseudogene on human chromosome 16 gi: 29124036 from nt coordinate 94647 to 95423 |
| Fig. 17: | Allignment of human chromosome 8 from nt coordinate 70000 to 72000 gi:21307426 with Human Cu/Zn processed pseudogene 71.4 gi:180712 |
| Fig. 18: | Alignment of human chromosome 8 (70000-72000) gi:21307426 with Human chromosome 21 gi 7768676 |
| Fig. 19: | Complete sequences of Cu/Zn processed pseudogene on human chromosome 8 gi :21307462, The 5’ and 3’ end (agaattgt), a 3’ poly (T) rich stretch and is flanked by short direct repeat sequences and TATTTAT( -29 ) upstream from the 5’ direct repeat are shown |
The 14 bases (from 70657 to 70670) which do not align with this SOD1 processed pseudogene was found to be a poly T sequence (Fig. 19).
Direct comparison of the complete DNA nucleotide sequences of the SOD1 Ψ 71.4 pseudogene on human chromosome 8 with the SOD1 functional gene on human chromosome 21 was carried out. The whole sequence of this gene has high identities with exon 2, 3, 4 (80, 94 and 84%, respectively) and partially with exon 5 (88%) and no alignment with exon 1 of SOD1 gene on human chromosome 21 (Fig. 18). The complete DNA nucleotide sequence (713 bases) of the Ψ 71.4 SOD1 pseudogene on human chromosome 8 is represented in Fig. 19.
DISCUSSION
NCBI-Blast analysis of the sequence alignment shows that the sequence of SOD1 amplified PCR product of genomic DNA and of SOD1 amplified cDNA are 97% identical. The amplicon of the cattle genomic DNA is 98% identical to mRNA of Bos taurus SOD1 sequences of the Gene Bank database. It also shares sequence homology with mRNA from other species such as Homo sapiens (87%), Capra hircus (95%) and Mucaca mulatto (91%). This indicates that this gene is an intronless.
The amplicon sequence of the native cattle genomic DNA aligns with four separate segments of Bos taurus chromosome 1 genomic. This findings indicates the presence of SOD1 normal gene i.e., gene with introns and exons on cattle chromosome BTA1. Superoxide dismutase 1 (SOD1) was mapped to cattle chromosome 1q12->q14 by in situ methods (Schmutz et al., 1996). It also shares sequence homology (99%) with Bos taurus chromosome BTA13 genomic similar to Superoxide dismutase. This indicates the presence of an intronless (processed) pseudogene on cattle chromosome BTA13. This finding agrees with Gallagher et al. (1999) who assigned bovine superoxide dismutase pseudogene to cattle chromosome BTA13. These results indicate that the gene under investigation is an intronless pseudogene. Pseudogenes fall into two distinct categories depending on the mechanism by which they are generated: Processed pseudogenes are reverse transcribed from mRNAs (and thus do not contain introns) whereas nonprocessed pseudogenes arise from duplications of genomic DNA (Mighell et al., 2000; Harrison et al., 2002a).
Four interesting alignments were found; alignment of the SOD1 amplicon cattle genomic DNA fragment showed 78% identities with human SOD1 processed pseudogene Ψ 69.1; 84% alignment with processed pseudogene Ψ 71.4; 80% alignment with human chromosome 16 DNA and 84% alignment with chromosome 8. The presence of SOD1 pseudogenes has been reported earlier by Danciger et al. (1986) who reported four functional processed pseudogenes not residing on chromosome 21. Three of them (69.1, 71.4 and Ψ J) originated from the 0.7-kilobase SOD1 mRNA, while the fourth (Ψ A1) was derived from the 0.9-kilobase mRNA species.
Study of the alignment of the two processed pseudogenes with chromosomes 8 and 16 reveals that processed pseudogene Ψ 69.1 (gi: 181206) is 94% identical with chromosome 16 and only 80% with chromosome 8. Whereas the processed pseudogene Ψ 71.4 (gi: 180712) is 98% identical with chromosome 8. These results indicate that SOD1 processed pseudogene Ψ 69.1 can be assigned to human chromosome 16 and SOD1 processed pseudogene Ψ 71.4 (gi: 180712) can be assigned to chromosome 8. Human chromosome 16 has been reported earlier to be associated with FALS (Sapp et al., 2003; Ruddy et al., 2003). As well as Messer et al. (1992) mapped the motor neuron degeneration gene in the mouse to proximal chromosome 8. He suggested that examination of human chromosome 8, which shows homology of synteny, in human kindreds with Familial Amyotrophic Lateral Sclerosis (FALS) as well as related hereditary neurologic diseases, might be fruitful. These results contradict a study carried out by Huret et al. (1987), using in situ hybridization which confirmed that SOD-1 gene localized in the segment enclosing the distal part of human chromosome 21 and no significant labeling on other chromosomes.
In order to identify the complete sequence of the SOD1 gene on chromosome 16, a 2000 bp (from nt coordinate 94000 to 96000) that comprises the human segment of chromosome 16 (from nt coordinate 94952 to 95227) is tested for alignment with the SOD1 processed pseudogenes (gi: 181206). Alignment with the processed pseudogene Ψ 69.1 (gi: 181206) shows that a segment 713 bp (from nt coordinate 94713 to 95423) has 94% alignment and a small fragment of 36 bp (from nt coordinate 94647 to 94682) has 100% alignment. The 30 bp (from nt coordinate 94683 to 94712) which does not align with this processed SOD1 pseudogene found to be poly A (Fig. 16). The results revealed that SOD1 retropseudogene is 777 bases. The sequence homology between the 2000 bp segment of human chromosome 16 and the SOD1 processed pseudogene Ψ 69.1 indicates that the SOD1 processed pseudogene Ψ 69.1 can be assigned to human chromosome 16. This agrees with the results of Sapp et al. (2003) that identified a putative locus on chromosome 16q12.
The whole sequence of retropseudogene on human chromosome 16 has identities with exon 2, 3, 4 (82, 86 and 82%, respectively) and partially with exon 5 (88%) and no alignment with exon 1 of SOD1 normal gene on human chromosome 21. This indicates that retropseudogene has many mutations such as deletions and base substitutions in its exons 2, 3, 4 and 5 whereas exon 1 is completely absent. Ruddy et al. (2003) suggested mutations of a gene on chromosome 16q12-13 are the significant cause of FALS. As well as Andersen et al. (2003) and Hough et al. (2004) stated that more than 90 point mutations in the SOD1 gene had been found to lead to the development of FALS. Kunst (2004) reported that the majority of mutations in SOD1 are missense mutations, with a small percentage of deletion and insertion mutations that result in prematurely terminated SOD1 polypeptides. The expression of a mutant SOD1 polypeptide, with or without residual SOD1 activity, is necessary to cause the FALS.
The complete sequence (777 nt) of SOD1 retropseudogene which is discovered and assigned on human chromosome 16 from 94647 nt to 95423 nt is represented in Fig. 16. This retropseudogene lacks introns, contains a 3’ poly (A)-rich stretch (30 nt). Retropseudogenes are typically characterized by a complete lake of intron, the presence of small flanking repeats and a polyadenine tail near the 3’ end (Zhang et al., 2003). Interestingly, it also contains 5’ (acgtc), 3’ (accgtc) direct repeats and TATA box (tataa) upstream from the 5’ direct repeat, in a position almost identical to the functional TATA box, indicating that it is intronless functional pseudogenes, supporting Danciger et al. (1986) who identified the processed pseudogenes Ψ 69.1 (gi: 181206) as a functional gene. These results also confirmed by Zhang and Gerstein (2004) who reported that in most cases, pseudogenes cannot produce transcripts as a result of functional promoter scarcity. Very rarely, some pseudogenes have either retained or acquired a functional promoter so they can be transcribed. Betràn et al. (2002) also suggested that there are a substantial number of processed genes of novel function that also derive from mRNAs of various intron-containing genes.
It was also possible to deduce the complete sequence of the SOD1 processed pseudogene on chromosome 8 (713 bp) from NCBI-Blast analysis of a 2000 nt segment of chromosome 8 with the complete processed pseudogene Ψ 71.4 (gi: 180712). The alignment shows that a 668 bp segment has 98% alignment and a small fragment 31 nt has 100% Identity. The 14 bp which does not align with this processed SOD1 pseudogene found to be poly T.
The whole sequence of the complete DNA nucleotide sequences of the SOD1 gene on human chromosome 8 has high identities with exon 2, 3, 4 and 5 and no alignment with exon 1 of SOD1 functional gene on human chromosome 21. Although the SOD1 processed pseudogene on human chromosome 8 overall sequence homology to the SOD1 coding region on human chromosome 21 was extensive, they contain multiple genetic lesions, such as deletions and base substitutions, that preclude the translation of the normal SOD1 polypeptide. It also bears the structural hallmarks of processed pseudogene; it lacks introns, the 5’ and 3’ direct repeats are presented. It also contains TATA box (TATTTAT) upstream from the 5’ direct repeat in a position almost identical to the functional TATA box (-29). It also contains a 3 poly (T)-rich stretch and is flanked by short direct repeat sequences. This results indicate that it is intronless functional pseudogenes, supporting Danciger et al. (1986) who identified the processed pseudogenes Ψ 71.4 (gi: 180712) as a functional gene and Nott et al. (2003) who reported that pseudogenes are often extremely conserved and transcriptionally active.
The presence of SOD1 functional retropseudoge on chromosome 16 and 8 reported in this study explains the reported FALS and other diseases in individuals with a normal chromosome 21.
ACKNOWLEDGMENTS
This work was partially supported by the executive programme on scientific and technological cooperation between the academy of scientific research and technology of Egypt and the Hungarian academy of sciences (Project: Natural immunity of cattle and other rural ruminants in Egypt). The author wishes to acknowledge Prof. Dr. Soheir M. El Nahas and Prof. Dr. Hanaa Abd El Sadek Oraby for their great effort in putting this manuscript in its final form and for their useful guidance and support.