This is the second installment in the quotes of interest series that focuses in particular on research and discussions from the 1980s, when noncoding DNA supposedly was ignored as irrelevant. The important message being offered is that there was plenty of research into possible functions or lack thereof in noncoding sequences of all types, and that whichever way authors concluded was based on the evidence available at the time, not ideology. This includes the parallel development of neutral theory, many proponents of which did conclude that pseudogenes were nonfunctional on the basis of their high mutation rates compared with coding sequences. Again, the point is not that no one argued against function (I argue against function at the organism level for most noncoding DNA), but that this is based on evidence, not unsupported assumption.
Members of the Alu family of interspersed repeated sequences and its rodent equivalents may be the normal cellular DNA replication initiation sites. In mammalian cells DNA replication proceeds bidirectionally simultaneously from many sites, and thus the initiation sites for replication might be expected to be interspersed repeated sequences with two-fold rotational symmetry. The inverted repeated examples of the Alu family of interspersed repeated sequences and their Chinese hamster equivalents show these attributes. These considerations raise the question of whether the transcription of these repeated sequences by RNA polymerase III, or the interaction of these sequences with the low molecular weight RNA, or both, may play a role in the initiation of DNA replication.
Jelinek, W.R., T.P. Toomey, L. Leinwand, C.H. Duncan, P.A. Biro, P.V. Choudary, S.M. Weissman, C.M. Rubin, C.M. Houck, P.L. Deininger, and C.W. Schmid. 1980. Ubiquitous, interspersed repeated sequences in mammalian genomes. Proceedings of the National Academy of Sciences of the USA 77: 1398-1402.
We have assigned six members of the human β-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned β-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome. Using intervening sequence probes, we showed that the functional gene is single copy and that all of the other β-actin related sequences are recently generated in evolution and are probably processed pseudogenes. The entire nucleotide sequence of the functional gene has been determined and is identical to cDNA clones in the coding and 5′ untranslated regions. We have previously reported that the 3′ untranslated region is well conserved between humans and rats (Ponte et al., Nucleic Acids Res. 12:1687-1696, 1984). Now we report that four additional noncoding regions are evolutionarily conserved, including segments of the 5′ flanking region, 5′ untranslated region, and, surprisingly, intervening sequences I and III. These conserved sequences, especially those found in the introns, suggest a role for internal sequences in the regulation of β-actin gene expression.
Our finding of highly conserved blocks of nucleotides in two of the five intervening sequences of β-acting genes raises the possibility that these segments have regulatory functions. Conserved internal regions have been reported previously, such as the internal transcriptional enhancer regions of immunoglobulin genes. However, the locations of these enhancers were initially regarded as a peculiarity of the immunoglobulin gene loci. More recently, internal control regions have been detected (but yet unidentified) for the adenovirus E1A gene, human globin genes, and chicken thymidine kinase gene. Any conclusion that the conserved β-actin intron sequences, especially those of IVS I, function as transcriptional enhancers must await direct experimentation. Nevertheless the evolutionary conservation of the immunoglobulin enhancer segments indicates that other transcriptional enhancers or cis-acting regulatory signals would be under selective pressure. It is interesting to note in this regard that the IVS I of both α- and β-globin genes are the most conserved introns of these genes. The IVS I of the human and mouse β-globin genes, for example, has 81 base pairs matching to give a KN(1) value of 0.302. Therefore these introns may well contain part of the proposed downstream regulatory elements.
Ng, S.-Y., P. Gunning, R. Eddy, P. Ponte, J. Leavitt, T. Shows, and L. Kedes. 1985. Evolution of the functional human β-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes. Molecular and Cellular Biology 5: 2720-2732.
Although the presence and similar location of pseudogenes in all the mammalian globin gene clusters suggest that pseudogenes may have some as yet unidentified function, the simplest explanation for their existence is that they are the natural consequence of the mechanisms of gene amplification and sequence divergence. The arrangement of genes within the human α-globin gene cluster is consistent with this possibility.
Proudfoot, N.J. and T. Maniatis. 1980. The structure of a human α-globin pseudogene and its relationship to α-globin gene duplication. Cell 21: 537-544.
In summary, the structural analysis of a number of different globin gene clusters suggests that globin gene families are in evolutionary flux. Perhaps pseudogenes are simply a natural consequence of the mechanisms by which multigene families evolve.
Lacy, E. and T. Maniatis. 1980. The nucleotide sequences of a rabbit β-globin pseudogene. Cell 21: 545-553.
Particularly surprising are the intron-exon splice borders of the H3.3 gene. Not only do they contain the standard splice consensus sequences, but in all cases the introns are flanked by 7-8 base pair direct repeats. The function, if any, of these repeats is unclear, since the repeats include both intron and exon bases. One functional difference between these introns can be inferred from the structures of the previously isolated cDNAs. Three of the cDNAs were shown to contain an unspliced intron, but did not carry introns 2 and 3. This could reflect the preferential splicing out of introns 2 and 3 before the splicing out of intron 1. If there is a tendency toward 5′ to 3′ splicing, the unusual splice junctions seen for the H3.3 gene could act to supersede this tendency. The advantage to the organism to remove intron 1 last is unclear but could point to some as yet undetermined function for this intron. In support of this, we have found that a DNA probe derived from intron 1 hybridizes to a single fragment in a Southern blot of total mouse genomic DNA indicating that the sequences in this intron may be conserved, whereas a DNA probe derived from intron 2 does not hybridize.
Wells, D., D. Hoffman, and L. Kedes. 1987. Unusual structure, evolutionary conservation of non-coding sequences and numerous pseudogenes characterize the human H3.3 histone multigene family. Nucleic Acids Research 15: 2871-2889.
A mouse α-globin-related pseudogene (ψα30.5) completely lacks intervening sequences, and could not code for a functional globin polypeptide because of frameshifts. The widespread occurrence of globin pseudogenes in other species suggests that they are not ‘dead’ genes but may be important in controlling globin expression.
The general hypothesis that pseudogenes control the productive genes in some fashion, nevertheless, remains attractive and we are investigating the hypothesis further, including tests in non-erythroid tissues. Certainly, the widespread occurrence of globin pseudogenes argues strongly for their functional importance.
Vanin, E.F., G.I. Goldberg, P.W. Tucker, and O. Smithies. 1980. A mouse α-globin-related pseudogene lacking intervening sequences. Nature 286: 222-226.
The foregoing data support the concept that the so-called “junk” or genetically inactive DNA centered around the centromeric region has a function in controlling the separation of centromere (or its replication into two daughter centromeres) at the junction of metaphase-anaphase in mitosis.
Vig, B.K. 1982. Sequence of centromere separation: role of centromeric heterochromatin. Genetics 102: 795-806.
A highly conserved repetitive DNA sequence, (TTAGGG)n, has been isolated from a human recombinant repetitive DNA library. Quantitative hybridization to chromosomes sorted by flow cytometry indicates that comparable amounts of this sequence are present on each human chromosome. Both fluorescent in situ hybridization and BAL-31 nuclease digestion experiments reveal major clusters of this sequence at the telomeres of all human chromosomes. The evolutionary conservation of this DNA sequence, its terminal chromosomal location in a variety of higher eukaryotes (regardless of chromosome number or chromosome length), and its similarity to functional telomeres isolated from lower eukaryotes suggest that this sequence is a functional human telomere.
The human genome contains a variety of DNA sequences present in multiple copies. These repetitive DNA sequences are thought to arise by many mechanisms, from direct sequence amplification to the unequal recombination of homologous DNA regions to the reverse flow of genetic information. While it is likely that some of these repetitive DNA sequences influence the structure and function of the human genome, little experimental evidence supports this idea at present.
We reasoned, however, that evolutionary conservation of a particular repetitive DNA sequence family might imply that the sequence is essential to cellular function.
Moyzis, R.K., J.M. Buckingham, L.S. Cram, M. Dani, L.L. Deaven, M.D. Jones, J. Meyne, R.L. Ratliff, and J.-R. Wu. 1988. A highly conserved repetitive DNA sequence, (TTAGGG)n, present in the telomeres of human chromosomes. Proceedings of the National Academy of Sciences of the USA 85: 6622-6626.
Part of the Quotes of interest series.