Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five “AD” missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from ρ° cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.     

Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification, and a novel form of polymorphism.

The human dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) gene family includes a functional gene (hDHFR) and at least four intronless genes. Three intronless genes (hDHFR-psi 2, hDHFR-psi 3, and hDHFR-psi 4) are identifiable as pseudogenes because of DNA sequence divergence from the functional gene with introns, while one intronless gene (hDHFR-psi 1) is completely homologous to the coding sequences of the functional gene. Analysis of genomic DNA from two panels of somatic human-rodent cell hybrids with specific molecular probes provide insight into the chromosomal organization and assignment of these genes. The five genes are dispersed in that each one is found on a different chromosome. The functional gene hDHFR has been assigned to chromosome 5, and one pseudogene (hDHFR-psi 4), to chromosome 3. In a human cell line (HeLa) that was selected for methotrexate resistance, the functional locus became amplified, while there was no amplification of the four intronless pseudogenes. hDHFR-psi 1 was found to be present in DNA of some individuals and absent from DNA of others, consistent with a recent evolutionary origin of this gene originally suggested by its sequence identity to the coding portions of the functional gene. The presence or absence of this intronless pseudogene represents a previously unreported form of DNA polymorphism.     

Are BET Inhibitors yet Promising Latency-Reversing Agents for HIV-1 Reactivation in AIDS Therapy?

AIDS first emerged decades ago; however, its cure, i.e., eliminating all virus sources, is still unachievable. A critical burden of AIDS therapy is the evasive nature of HIV-1 in face of host immune responses, the so-called “latency.” Recently, a promising approach, the “Shock and Kill” strategy, was proposed to eliminate latently HIV-1-infected cell reservoirs. The “Shock and Kill” concept involves two crucial steps: HIV-1 reactivation from its latency stage using a latency-reversing agent (LRA) followed by host immune responses to destroy HIV-1-infected cells in combination with reinforced antiretroviral therapy to kill the progeny virus. Hence, a key challenge is to search for optimal LRAs. Looking at epigenetics of HIV-1 infection, researchers proved that some bromodomains and extra-terminal motif protein inhibitors (BETis) are able to reactivate HIV-1 from latency. However, to date, only a few BETis have shown HIV-1-reactivating functions, and none of them have yet been approved for clinical trial. In this review, we aim to demonstrate the epigenetic roles of BETis in HIV-1 infection and HIV-1-related immune responses. Possible future applications of BETis and their HIV-1-reactivating properties are summarized and discussed.     

Long-range disruption of gene expression by a selectable marker cassette

Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.     

The olfactory receptor gene repertoire in primates and mouse: evidence for reduction of the functional fraction in primates

Olfactory receptors (ORs) located in the cell membrane of olfactory sensory neurons of the nasal epithelium are responsible for odor detection by binding specific odorant ligands. Primates are thought to have a reduced sense of smell (microsmatic) with respect to other mammals such as dogs or rodents. We have previously demonstrated that over 70% of the human OR genes have become nonfunctional pseudogenes, leading us to hypothesize that the reduced sense of smell could correlate with the loss of functional genes. To extend these results, we sampled the OR gene repertoire of 10 primate species, from prosimian lemur to human, in addition to mouse. About 221 previously unidentified primate sequences and 33 mouse sequences were analyzed. These sequences encode ORs distributed in seven families and 56 subfamilies. Analysis showed a high fraction ( approximately 50% on average) of pseudogenes in hominoids. In contrast, only approximately 27% of OR genes are pseudogenes in Old World monkeys, and New World monkeys are almost free of pseudogenes. The prosimian branch seems to have evolved differently from the other primates and has approximately 37% pseudogene content. No pseudogenes were found in mouse. With the exception of New World monkeys, we demonstrate that primates have a high fraction of OR pseudogenes compared with mouse. We hypothesize that under relaxed selective constraints, primates would have progressively accumulated pseudogenes with the highest level seen in hominoids. The fraction of pseudogenes in the OR gene repertoire could parallel the evolution of the olfactory sensory function.     

Mouse alpha-globin genes and alpha-globin-like pseudogenes are not syntenic.

A genetic polymorphism for a Bgl I endonuclease site near the alpha-globin-like pseudogene alpha-4 of C57BL/6 and C3H/HeN mice was used to show that alpha-4 was not affected by three independent mutations in which the adult globin genes alpha-1 and alpha-2 were deleted. These results indicated that alpha-4 might not be located adjacent to the adult alpha-globin genes on chromosome 11. Restriction endonuclease analysis of DNA of a primary clone of a Chinese hamster--mouse somatic cell hybrid that had lost mouse chromosomes 11 and 18 showed that this clone lacked the adult murine globin genes alpha-1 and alpha-2 but it did contain the alpha-globin-like pseudogenes alpha-3 and alpha-4. These results indicated that the adult alpha-globin genes and alpha-globin-like pseudogenes are not located on the same chromosome. Similar analyses of several other Chinese hamster--mouse somatic cell hybrids that had segregated other mouse chromosomes indicated that the alpha-globin-like pseudogenes alpha-3 and alpha-4 are located on mouse chromosomes 15 and 17, respectively. These data explain why alpha-3 and alpha-4 were not affected by the three independently induced deletion-type mutations that cause alpha-thalassemia in the mouse.     

CCR5 as a Natural and Modulated Target for Inhibition of HIV

Human immunodeficiency virus type 1 (HIV-1) infection of target cells requires CD4 and a co-receptor, predominantly the chemokine receptor CCR5. CCR5-delta32 homozygosity results in a truncated protein providing natural protection against HIV infection—this without detrimental effects to the host—and transplantation of CCR5-delta32 stem cells in a patient with HIV (“Berlin patient”) achieved viral eradication. As a more feasible approach gene-modification strategies are being developed to engineer cellular resistance to HIV using autologous cells. We have developed a dual therapeutic anti-HIV lentiviral vector (LVsh5/C46) that down-regulates CCR5 and inhibits HIV-1 fusion via cell surface expression of the gp41-derived peptide, C46. This construct, effective against multiple strains of both R5- and X4-tropic HIV-1, is being tested in Phase I/II trials by engineering HIV-resistant hematopoietic cells.     

Challenges in the Design of a T Cell Vaccine in the Context of HIV-1 Diversity

The extraordinary variability of HIV-1 poses a major obstacle to vaccine development. The effectiveness of a vaccine is likely to vary dramatically in different populations infected with different HIV-1 subtypes, unless innovative vaccine immunogens are developed to protect against the range of HIV-1 diversity. Immunogen design for stimulating neutralizing antibody responses focuses on “breadth” – the targeting of a handful of highly conserved neutralizing determinants on the HIV-1 Envelope protein that can recognize the majority of viruses across all HIV-1 subtypes. An effective vaccine will likely require the generation of both broadly cross-neutralizing antibodies and non-neutralizing antibodies, as well as broadly cross-reactive T cells. Several approaches have been taken to design such broadly-reactive and cross-protective T cell immunogens. Artificial sequences have been designed that reduce the genetic distance between a vaccine strain and contemporary circulating viruses; “mosaic” immunogens extend this concept to contain multiple potential T cell epitope (PTE) variants; and further efforts attempt to focus T cell immunity on highly conserved regions of the HIV-1 genome. Thus far, a number of pre-clinical and early clinical studies have been performed assessing these new immunogens. In this review, the potential use of these new immunogens is explored.     

Human cytomegalovirus IE1 and IE2 proteins are mutagenic and mediate “hit-and-run” oncogenic transformation in cooperation with the adenovirus E1A proteins

Some epidemiological studies have suggested a possible link between human cytomegalovirus (HCMV) infection and various malignancies, and HCMV has been shown to transform cultured cells. However, viral DNA is not detected in most transformants, and the mechanism by which HCMV might contribute to oncogenesis has remained obscure. Here we show that the HCMV immediate early 1 and 2 genes can cooperate with the adenovirus E1A gene to generate transformed foci of primary baby rat kidney cells. HCMV gene expression is transient and viral DNA is not present in clonal cell lines derived from the transformed foci. We find that the HCMV immediate early proteins are mutagenic, and we propose that HCMV has the potential to contribute to oncogenesis through a “hit-and-run” mechanism, by inducing mutations in cellular genes.