A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.

High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas.

Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2-encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene.

High frequency of sex and equal frequencies of mating types in natural populations of the ciliate Tetrahymena thermophila.

In ciliate protists, sex involves the temporary joining of two cells of compatible mating type, followed by meiosis and exchange of gametic nuclei between conjugants. Reproduction is by asexual binary fission following conjugation. For the many ciliates with fixed multiple mating types, frequency-dependent sex-ratio theory predicts equal frequencies of mating types, if sex is common in nature. Here, we report that in natural populations of Tetrahymena thermophila sexually immature cells, indicative of recent conjugation, are found from spring through fall. In addition, the seven mating types occur in approximately equal frequencies, and these frequencies appear to be maintained by interaction between complex, multiple mat alleles and environmental conditions during conjugation. Such genotype-environment interaction determining mating type frequency is rare among ciliates.

Social transmission of reproductive behavior increases frequency of inherited disorders in a young-expanding population

The observation of high frequencies of certain inherited disorders in the population of Saguenay–Lac Saint Jean can be explained in terms of the variance and the correlation of effective family size (EFS) from one generation to the next. We have shown this effect by using the branching process approach with real demographic data. When variance of EFS is included in the model, despite its profound effect on mutant allele frequency, any mutant introduced in the population never reaches the known carrier frequencies (between 0.035 and 0.05). It is only when the EFS correlation between generations is introduced into the model that we can explain the rise of the mutant alleles. This correlation is described by a c parameter that reflects the dependency of children’s EFS on their parents’ EFS. The c parameter can be considered to reflect social transmission of demographic behavior. We show that such social transmission dramatically reduces the effective population size. This could explain particular distributions in allele frequencies and unusually high frequency of certain inherited disorders in some human populations.

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N × Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH (≈38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.

Double strand breaks at the HIS2 recombination hot spot in Saccharomyces cerevisiae

Double strand breaks (DSBs) have been found at several meiotic recombination hot spots in Saccharomyces cerevisiae; more global studies have found that they occur at many places along several yeast chromosomes during meiosis. Indeed, the number of breaks found is consistent with the number of recombination events predicted from the genetic map. We have previously demonstrated that the HIS2 gene is a recombination hot spot, exhibiting a high frequency of gene conversion and associated crossing over. This paper shows that DSBs occur in meiosis at a site in the coding region and at a site downstream of the HIS2 gene and that the DSBs are dependent upon genes required for recombination. The frequency of DSBs at HIS2 increases when the gene conversion frequency is increased by alterations in the DNA around HIS2, and vice versa. A deletion that increases both DSBs and conversion can stimulate both when heterozygous; that is, it is semidominant and acts to stimulate DSBs in trans. These data are consistent with the view that homologous chromosomes associate with each other before the formation of the DSBs.

Duplicated genes evolve independently after polyploid formation in cotton

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

Thalamocortical dysrhythmia: A neurological and neuropsychiatric syndrome characterized by magnetoencephalography

Spontaneous magnetoencephalographic activity was recorded in awake, healthy human controls and in patients suffering from neurogenic pain, tinnitus, Parkinson's disease, or depression. Compared with controls, patients showed increased low-frequency θ rhythmicity, in conjunction with a widespread and marked increase of coherence among high- and low-frequency oscillations. These data indicate the presence of a thalamocortical dysrhythmia, which we propose is responsible for all the above mentioned conditions. This coherent θ activity, the result of a resonant interaction between thalamus and cortex, is due to the generation of low-threshold calcium spike bursts by thalamic cells. The presence of these bursts is directly related to thalamic cell hyperpolarization, brought about by either excess inhibition or disfacilitation. The emergence of positive clinical symptoms is viewed as resulting from ectopic γ-band activation, which we refer to as the “edge effect.” This effect is observable as increased coherence between low- and high-frequency oscillations, probably resulting from inhibitory asymmetry between high- and low-frequency thalamocortical modules at the cortical level.

Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: Toward a genetic model of meningioma progression

Nineteen benign [World Health Organization (WHO) grade I; MI], 21 atypical (WHO grade II; MII), and 19 anaplastic (WHO grade III; MIII) sporadic meningiomas were screened for chromosomal imbalances by comparative genomic hybridization (CGH). These data were supplemented by molecular genetic analyses of selected chromosomal regions and genes. With increasing malignancy grade, a marked accumulation of genomic aberrations was observed; i.e., the numbers (mean ± SEM) of total alterations detected per tumor were 2.9 ± 0.7 for MI, 9.2 ± 1.2 for MII, and 13.3 ± 1.9 for MIII. The most frequent alteration detected in MI was loss on 22q (58%). In MII, aberrations most commonly identified were losses on 1p (76%), 22q (71%), 14q (43%), 18q (43%), 10 (38%), and 6q (33%), as well as gains on 20q (48%), 12q (43%), 15q (43%), 1q (33%), 9q (33%), and 17q (33%). In MIII, most of these alterations were found at similar frequencies. However, an increase in losses on 6q (53%), 10 (68%), and 14q (63%) was observed. In addition, 32% of MIII demonstrated loss on 9p. Homozygous deletions in the CDKN2A gene at 9p21 were found in 4 of 16 MIII (25%). Highly amplified DNA sequences were mapped to 12q13–q15 by CGH in 1 MII. Southern blot analysis of this tumor revealed amplification of CDK4 and MDM2. By CGH, DNA sequences from 17q were found to be amplified in 1 MII and 8 MIII, involving 17q23 in all cases. Despite the high frequency of chromosomal aberrations in the MII and MIII investigated, none of these tumors showed mutations in exons 5–8 of the TP53 gene. On the basis of the most common aberrations identified in the various malignancy grades, a model for the genomic alterations associated with meningioma progression is proposed.