Association of a truncated cytochrome c processed pseudogene with a similarly truncated member from a long interspersed repeat family of rat.

The cytochrome c multigene family of rat contains approximately 30 processed pseudogenes that represent genomic DNA copies of three alternate mRNAs. Here, the DNA sequence of an unusual processed pseudogene reveals that it has a complete 3' noncoding region including a short poly A tail but unlike the others is abruptly truncated at its 5' end, 19 amino acid codons from the translation terminator. At this position the pseudogene is fused through 17 consecutive adenylic acid residues to a 1.3 kb repetitive sequence. This repetitive element is flanked by direct repeats and represents a truncated member from a major long interspersed repeat family. The rat element is a composite of sequences observed in long interspersed repeats from both rodents and primates. Comparison to the equivalent mouse sequences shows that the 5' half of the repeat distal to the pseudogene has an open reading frame and is highly conserved whereas the half adjacent to the pseudogene is evolutionarily unstable. The proportion of cytochrome c pseudogene recombinant clones containing this repetitive DNA is 3 fold greater than observed in random isolates and may reflect a general tendency of processed pseudogenes to associate with other repetitive sequences in the genome.     

Genes and pseudogenes for human U2 RNA: Implications for the mechanism of pseudogene formation

Three loci, designated U2/4, U2/6 and U2/7, which contain sequences related to human U2 RNA, have been studied. The U2/6 locus contains a tandem array of bona fide U2 genes. U2/4 and U2/7, in contrast, contain pseudogenes whose sequences deviate significantly from that of mammalian U2 RNA. The two pseudogenes appear to have been created by different mechanisms. The sequences that flank the pseudogene in the U2/4 locus lack homology to the corresponding sequences in functional human U2 genes, except for 10 base-pairs immediately following the 3′ end. The conserved 3′-flanking segment is homologous to those nucleotides that are present in a U2 RNA precursor. No direct repeats flank the pseudogene in the U2/4 locus. The observations thus suggest that a complementary DNA copy of the U2 RNA precursor was inserted into a blunt-ended chromosomal break to generate the U2/4 locus.The U2/7 locus, in contrast, revealed flanking sequence homology when compared to functional U2 genes, both on the 5′ and 3′ sides of the pseudogene. The homology was interrupted on both sides by repetitive sequences belonging to the Alu family. On the 5′ side the homology continues beyond the Alu repeats whereas on the 3′ side it ends precisely at the Alu repeat. This Alu repeat is inserted in a region where a homocopolymeric region of alternating C and T residues is located in functional U2 loci. The observed organization of the U2/7 locus suggests that a previously functional U2 locus was invaded by Alu repeats and subsequently accumulated base substitutions to become a pseudogene.     

Genomic expansion of Marek''s disease virus DNA is associated with serial in vitro passage.

An EcoRI restriction endonuclease pattern of Md11 virus DNA, a very virulent strain of Marek's disease virus (MDV), was obtained by using total cellular DNA from infected cells. With the EcoRI restriction endonuclease pattern and a published BamHI map of MDV (Fukuchi et al., J. Virol. 51:102-109), we constructed a partial EcoRI map of a series of MDV clones (gift from H. J. Kung). The clones were used to identify a region of the Md11 genome which is altered as the oncogenic virus is passaged in vitro. This region was mapped into a 1.8-kilobase segment in the inverted-repeat sequences flanking the long unique region of the virus genome. The alteration appeared to result from multiple DNA insertions that produced an increase of 0.6 to 5.4 kilobases. Although the expansion of this region did not diminish the ability of MDV to replicate in vitro, it may be associated with the loss of Marek's disease oncogenicity.     

Locations of three repetitive sequence families found in BALB/c adult beta-globin clones.

Three different repeat sequences have been mapped within the cloned EcoRI fragments that contain the adult beta-globin genes from the BALB/c (Hddd) mouse. One sequence, "a", occurs 1.5-2 kb 3' to the beta-major gene. A second, "b", is found 4kb 5' and 7.5kb 3' to the beta-minor gene. The 14kb EcoRI fragment bearing the beta-minor gene carries at least one additional repetitive element, "c". Probing a BALB/c DNA library with each repeat has demonstrated that these sequences are moderately to highly repetitive and are extensively interspersed with each other throughout the genome. In addition, repeats "a" and "b" are preferentially found in satellite and main-band DNa, respectively. The occurrence of these repeats elsewhere in the beta-globin cluster was demonstrated by probing the non-adult globin clones with each repeat. The arrangement of these repeats around the non-adult genes is 5'-"b"-"b"-epsilon y-beta hl-beta h2-"c"-beta h3-3'. Probing the C57BL/10 (Hbbs) adult gene clones with these repeats demonstrated that the distribution of these sequences in the adult region of these two haplotypes is essentially the same.     

Nonrandom integration of human U4 RNA pseudogenes.

Four loci for human U4 RNA have been characterized by DNA sequence analysis. The results show that all four loci represent pseudogenes, which are flanked by direct repeats. Three of the pseudogenes, designated U4/5, U4/6, and U4/8, have very similar structures; they are all truncated and contain the first 67 to 68 nucleotides of the U4 RNA sequence. Their properties suggest that they were created by integration of truncated cDNA copies of the U4 RNA into new chromosomal sites. An interesting observation was that their flanking regions exhibit sequence homology. A purine-rich 5'-flanking sequence 12 to 13 nucleotides long is almost perfectly conserved in all three loci. Boxes of homology were also found on the 3' side when the U4/6 and U4/8 loci were compared. The U4/4 locus has a slightly different structure; the pseudogene matches the first 79 nucleotides of U4 RNA, but contains a greater number of mutations than the other pseudogenes. Taken together, the results suggest that a frequently occurring type of pseudogene for human U4 was created by a RNA-mediated mechanism and that the integration sites have features in common.     

Novel structure of a human U6 snRNA pseudogene

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.     

Methylation and rearrangement of mouse intracisternal a particle genes in development, aging, and myeloma.

Sequences of DNA that hybridize on Southern blots with cloned intracisternal A-particle (IAP) sequences have been examined in genomic DNAs of neonatal mice, livers of adult mice (3, 6, 12, 18, 24, and 26 months old), and the solid myeloma tumor MOPC-315. The isoschizomers HpaII (CCGG or mCCGG) and MspI (CCGG or CmCGG) were used to assess methylation. All the DNAs produced a major 0.5-kilobase MspI fragment that hybridizes with IAP probe. Only the myeloma DNA, and to a much lesser degree DNA from senescent mouse liver, produced this fragment in HpaII digest; the other DNAs all had IAP sequences resistant to HpaII digestion. These sequences thus become fully methylated to CmCGG early and remain so in adult life, except in the myeloma cells that are expressing the IAP genes. An increase in MspI-sensitive sites in IAP gene-containing DNA was observed in aging mice. The probe used to assess methylation, a 0.8-kilobase fragment produced by BamHI-HindIII double digestion, is common to several cloned IAP genes and is part of a region of DNA which is conserved in genomes of all mouse tissues. The probe hybridized to 1.5- and 1.4-kilobase doublet bands produced by BamHI, HindIII, and EcoRI triple digestions of neonatal DNA. These two bands were found in neonatal livers of Swiss Webster, BALB/c, and C57BL/6J mouse strains, showed less in adult liver, and were barely detectable in senescent livers from C57BL/6J mice.     

RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3′-UTR的siRNA沉默HUTP14C mRNA后,再用RT PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3′-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3′-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。     

Molecular Nature of Spontaneous Mutations in Mouse Lactate Dehydrogenase-a Processed Pseudogenes

The presence of at least ten mouse LDH-A pseudogenes was demonstrated in the genomic blot analysis, and four different processed pseudogenes have thus far been isolated and characterized. In this report, the nucleotide sequences to two different mouse lactate dehydrogenase-A processed pseudogenes, M11 and M14, were determined and compared with the protein-coding sequences of the mouse and rat LDH-A functional genes. In the pseudogene M11, the sequence of 64 nucleotides from codon no. 257 to 278 was tandemly duplicated. In the pseudogene M14, the sequence of 22 nucleotides from codon no. 68 to 75 was replaced by an inserted repetitive sequence of 242 nucleotides homologous to a mouse truncated R element. The pattern of nucleotide substitutions accumulated in mouse LDH-A pseudogenes M11 and M14, as well as that of pseudogene M10 identified previously, was analyzed, and the substitution frequencies of the C or G at the CG dinucleotide were found to be high.     

Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket,Acheta domesticus (Orthoptera:Gryllidae)

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.     

Evolution of human cytoplasmic actin gene sequences: chromosome mapping and structural characterizations of three cytoplasmic actin-like pseudogenes including one on the Y chromosome

Eight recombinant phage clones containing cytoplasmic actin-like gene sequences have been isolated from a human genomic library for structural characterization. Kpn I family repeat sequences flank six of these actin genes isolated, and Alu family repeats are scattered throughout the DNA inserts of all eight phage clones. Three of these genes are γ actin-like, and the other five are β actin-like. The complete nucleotide sequence analysis of one β and one γ actin-like genes and their flanking regions demonstrates that they both are processed pseudogenes. Using unique DNA sequences flanking these two pseudogenes as hybridization probes for human-mouse somatic cell hybrid DNAs, we have mapped the two actin pseudogenes on human chromosomes 8 and 3, respectively. We have also determined the DNA sequence of a human Y chromosome-linked, processed actin pseudogene. The different values of sequence divergence of these processed pseudogenes and their functional counterparts allow us to estimate the time of generation of the pseudogenes. The results suggest that the cDNA insertion events generating the human cytoplasmic actin-like pseudogenes have occurred at significantly different times during the evolution of primates, after their separation from other mammalian species.     

An expressed beta-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed beta-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human x Chinese hamster somatic cell hybrids cleaved with Hind III or EcoRI. Probes containing the 3' untranslated regions of the expressed gene M40 and of pseudogene 21 beta were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21----6pter, the 21 beta pseudogene (TUBBP1) to chromosome 8 region 8q21----8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

Mouse cytoskeletal gamma-actin: analysis and implications of the structure of cloned cDNA and processed pseudogenes

The nucleotide sequence corresponding to almost the whole of a mouse gamma-cytoskeletal actin mRNA was determined from overlapping cloned DNA copies derived from brain mRNA. Several gamma-actin processed pseudogenes were isolated from a library of cloned DBA mouse genomic DNA, and the nucleotide sequences of these were determined and compared with that of the cDNA. This showed that two of these pseudogenes had arisen from a gene duplication or amplification event, and indicated that they had subsequently undergone partial correction against one another. The relative ages of the pseudogenes were estimated on the basis of their percentage divergence from the cDNA sequence and these were compared with an estimation based on the number of presumed silent mutations in the cDNA since each pseudogene had arisen. Consistent results were obtained, except in the case of one pseudogene which also showed an anomalous regional distribution of differences from the cDNA sequence. One way of accounting for the features of this anomalous pseudogene is by postulating that it is derived from a second functional gene for gamma-actin, different from that represented by the cDNA described here.     

Amplified ribosomal spacer sequence: structure and evolutionary origin

A novel class of repeated sequences consisting of tandem arrays of ribosomal spacer sequence has been discovered in a mouse genome. Comparison to normal ribosomal DNA reveals that one repeat unit consists of two separate parts of spacer sequence. This amplified spacer sequence has a pseudogene-like structure but is distinct from the previously reported pseudogenes and orphons in regions lacking coding sequences. So far the amplified spacer sequence has been found only in the BALB/c mouse genome but not in ten other laboratory strains and several wild-type mouse stocks. Surprisingly, a part of the amplified spacer sequence unit had a higher homology to the corresponding part of the ribosomal DNA sequence of Mus musculus molossinus, a Japanese wild-type mouse, than to the corresponding part of the rDNA of the BALB/c mouse. These findings suggest that the amplified spacer sequence of the BALB/c mouse might have partly originated in M. m. molossinus or in a related subspecies.     

A marsupial phosphoglycerate kinase (PGK) processed pseudogene

A clone that cross-hybridized with a full-length human cDNA PGK probe was isolated from a hill kangaroo (Macropus robustus: Marsupialia) lambda EMBL4 EcoRI genomic library. The clone was sequenced and demonstrated to be a pseudogene, with two deletions (one of 3 bases, the other 24 bases long), one single base insertion, and a nonsense mutation with respect to the functional human X-linked gene. It is flanked by terminal repeats in the 5' and 3' noncoding regions, but it has no 3' poly(A) remnant. The 3' untranslated region has a 34-bp sequence, with 29 bp homologous to the human 3' untranslated region. The overall percentage homology with the mouse and human X-linked PGK indicates that this pseudogene is probably more closely related to eutherian X-linked PGK genes than to the autosomal form. The results also suggest that pseudogenes are of considerable antiquity (greater than 100 MYr) in the mammalian lineage.