Extent of genomic rearrangement after genome duplication in yeast

Whole-genome duplication approximately 108 years ago was proposed as an explanation for the many duplicated chromosomal regions in Saccharomyces cerevisiae. Here we have used computer simulations and analytic methods to estimate some parameters describing the evolution of the yeast genome after this duplication event. Computer simulation of a model in which 8% of the original genes were retained in duplicate after genome duplication, and 70–100 reciprocal translocations occurred between chromosomes, produced arrangements of duplicated chromosomal regions very similar to the map of real duplications in yeast. An analytical method produced an independent estimate of 84 map disruptions. These results imply that many smaller duplicated chromosomal regions exist in the yeast genome in addition to the 55 originally reported. We also examined the possibility of determining the original order of chromosomal blocks in the ancestral unduplicated genome, but this cannot be done without information from one or more additional species. If the genome sequence of one other species (such as Kluyveromyces lactis) were known it should be possible to identify 150–200 paired regions covering the whole yeast genome and to reconstruct approximately two-thirds of the original order of blocks of genes in yeast. Rates of interchromosome translocation in yeast and mammals appear similar despite their very different rates of homologous recombination per kilobase.

Maize as a model for the evolution of plant nuclear genomes

The maize genome is replete with chromosomal duplications and repetitive DNA. The duplications resulted from an ancient polyploid event that occurred over 11 million years ago. Based on DNA sequence data, the polyploid event occurred after the divergence between sorghum and maize, and hence the polyploid event explains some of the difference in DNA content between these two species. Genomic rearrangement and diploidization followed the polyploid event. Most of the repetitive DNA in the maize genome is retrotransposable elements, and they comprise 50% of the genome. Retrotransposon multiplication has been relatively recent—within the last 5–6 million years—suggesting that the proliferation of retrotransposons has also contributed to differences in DNA content between sorghum and maize. There are still unanswered questions about repetitive DNA, including the distribution of repetitive DNA throughout the genome, the relative impacts of retrotransposons and chromosomal duplication in plant genome evolution, and the hypothesized correlation of duplication events with transposition. Population genetic processes also affect the evolution of genomes. We discuss how centromeric genes should, in theory, contain less genetic diversity than noncentromeric genes. In addition, studies of diversity in the wild relatives of maize indicate that different genes have different histories and also show that domestication and intensive breeding have had heterogeneous effects on genetic diversity across genes.

Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias

Panhandle PCR amplifies genomic DNA with known 5′ and unknown 3′ sequences from a template with an intrastrand loop schematically shaped like a pan with a handle. We used panhandle PCR to clone MLL genomic breakpoints in two pediatric treatment-related leukemias. The karyotype in a case of treatment-related acute lymphoblastic leukemia showed the t(4;11)(q21;q23). Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5′ MLL breakpoint cluster region (bcr). The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single MLL gene rearrangement. Panhandle PCR amplified the breakpoint at position 1493 in MLL intron 6. Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of MLL intron 6 with a previously uncharacterized sequence in MLL intron 1, consistent with a partial duplication. In both cases, the breakpoints in the MLL bcr were in Alu repeats, and there were Alu repeats in proximity to the breakpoints in the partner DNAs, suggesting that Alu sequences were relevant to these rearrangements. This study shows that panhandle PCR is an effective method for cloning MLL genomic breakpoints in treatment-related leukemias. Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3′ distribution in the bcr that has been found in adult cases.

Spectral karyotyping suggests additional subsets of colorectal cancers characterized by pattern of chromosome rearrangement

The abundant chromosome abnormalities in most carcinomas are probably a reflection of genomic instability present in the tumor, so the pattern and variability of chromosome abnormalities will reflect the mechanism of instability combined with the effects of selection. Chromosome rearrangement was investigated in 17 colorectal carcinoma-derived cell lines. Comparative genomic hybridization showed that the chromosome changes were representative of those found in primary tumors. Spectral karyotyping (SKY) showed that translocations were very varied and mostly unbalanced, with no translocation occurring in more than three lines. At least three karyotype patterns could be distinguished. Some lines had few chromosome abnormalities: they all showed microsatellite instability, the replication error (RER)+ phenotype. Most lines had many chromosome abnormalities: at least seven showed a surprisingly consistent pattern, characterized by multiple unbalanced translocations and intermetaphase variation, with chromosome numbers around triploid, 6–16 structural aberrations, and similarities in gains and losses. Almost all of these were RER−, but one, LS411, was RER+. The line HCA7 showed a novel pattern, suggesting a third kind of genomic instability: multiple reciprocal translocations, with little numerical change or variability. This line was also RER+. The coexistence in one tumor of two kinds of genomic instability is to be expected if the underlying defects are selected for in tumor evolution.

The role of genetic and genomic attributes in the success of polyploids

In 1950, G. Ledyard Stebbins devoted two chapters of his book Variation and Evolution in Plants (Columbia Univ. Press, New York) to polyploidy, one on occurrence and nature and one on distribution and significance. Fifty years later, many of the questions Stebbins posed have not been answered, and many new questions have arisen. In this paper, we review some of the genetic attributes of polyploids that have been suggested to account for the tremendous success of polyploid plants. Based on a limited number of studies, we conclude: (i) Polyploids, both individuals and populations, generally maintain higher levels of heterozygosity than do their diploid progenitors. (ii) Polyploids exhibit less inbreeding depression than do their diploid parents and can therefore tolerate higher levels of selfing; polyploid ferns indeed have higher levels of selfing than do their diploid parents, but polyploid angiosperms do not differ in outcrossing rates from their diploid parents. (iii) Most polyploid species are polyphyletic, having formed recurrently from genetically different diploid parents. This mode of formation incorporates genetic diversity from multiple progenitor populations into the polyploid “species”; thus, genetic diversity in polyploid species is much higher than expected by models of polyploid formation involving a single origin. (iv) Genome rearrangement may be a common attribute of polyploids, based on evidence from genome in situ hybridization (GISH), restriction fragment length polymorphism (RFLP) analysis, and chromosome mapping. (v) Several groups of plants may be ancient polyploids, with large regions of homologous DNA. These duplicated genes and genomes can undergo divergent evolution and evolve new functions. These genetic and genomic attributes of polyploids may have both biochemical and ecological benefits that contribute to the success of polyploids in nature.

Genomic evolution during a 10,000-generation experiment with bacteria

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

Transient excess of MYC activity can elicit genomic instability and tumorigenesis

Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been attributed to sustained effects on proliferation and differentiation. Here we report that MYC may also contribute to tumorigenesis by destabilizing the cellular genome. A transient excess of MYC activity increased tumorigenicity of Rat1A cells by at least 50-fold. The increase persisted for >30 days after the return of MYC activity to normal levels. The brief surfeit of MYC activity was accompanied by evidence of genomic instability, including karyotypic abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts, although these cells did not become tumorigenic. Stimulation of Rat1A cells with MYC accelerated their passage through G1/S. Moreover, MYC could force normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-l-aspartate (PALA) at concentrations that normally lead to arrest in S phase by checkpoint mechanisms. Instead, the cells subsequently appeared to arrest in G2. We suggest that the accelerated passage through G1 was mutagenic but that the effect of MYC permitted a checkpoint response only after G2 had been reached. Thus, MYC may contribute to tumorigenesis through a dominant mutator effect.

Genetic analysis using genomic representations

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible “representations” of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia

The TEL (ETV6)−AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in ≈25% of the most predominant subtype of leukemia— common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia.

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

The 2.0-Å resolution crystal structure of a trimeric antibody fragment with noncognate VH–VL domain pairs shows a rearrangement of VH CDR3

The 2.0-Å resolution x-ray crystal structure of a novel trimeric antibody fragment, a “triabody,” has been determined. The trimer is made up of polypeptides constructed in a manner identical to that previously described for some “diabodies”: a VL domain directly fused to the C terminus of a VH domain—i.e., without any linker sequence. The trimer has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. For the particular structure reported here, the polypeptide was constructed with a VH domain from one antibody fused to the VL domain from an unrelated antibody giving rise to “combinatorial” Fvs upon formation of the trimer. The structure shows that the exchange of the VL domain from antibody B1-8, a Vλ domain, with the VL domain from antibody NQ11, a Vκ domain, leads to a dramatic conformational change in the VH CDR3 loop of antibody B1-8. The magnitude of this change is similar to the largest of the conformational changes observed in antibody fragments in response to antigen binding. Combinatorial pairing of VH and VL domains constitutes a major component of antibody diversity. Conformationally flexible antigen-binding sites capable of adapting to the specific CDR3 loop context created upon VH–VL pairing may be employed by the immune system to maximize the structural diversity of the immune response.

Terpenoid biosynthesis from 1-deoxy-d-xylulose in higher plants by intramolecular skeletal rearrangement

The incorporation of [1-13C]- and [2,3,4,5-13C4]1-deoxy-d-xylulose into β-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13C Enrichment and 13C13C coupling patterns showed conclusively that 1-deoxy-d-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, β-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

Genomic analysis of human and mouse TCL1 loci reveals a complex of tightly clustered genes

TCL1 and TCL1b genes on human chromosome 14q23.1 are activated in T cell leukemias by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T cell receptor genes. In this report we present the cloning, mapping, and expression analysis of the human and murine TCL1/Tcl1 locus. In addition to TCL1 and TCL1b, the human locus contains two additional genes, TCL1-neighboring genes (TNG) 1 and 2, encoding proteins of 141 and 110 aa, respectively. Both genes show no homology to any known genes, but their expression profiles are very similar to those of TCL1 and TCL1b. TNG1 and TNG2 also are activated in T cell leukemias with rearrangements at 14q32.1. To aid in the development of a mouse model we also have characterized the murine Tcl1 locus and found five genes homologous to human TCL1b. Tcl1b1–Tcl1b5 proteins range from 117 to 123 aa and are 65–80% similar, but they show only a 30–40% similarity to human TCL1b. All five mouse Tcl1b and murine Tcl1 mRNAs are abundant in mouse oocytes and two-cell embryos but rare in various adult tissues and lymphoid cell lines. These data suggest a similar or complementary function of these proteins in early embryogenesis.

A model system to study genomic imprinting of human genes

Somatic-cell hybrids have been shown to maintain the correct epigenetic chromatin states to study developmental globin gene expression as well as gene expression on the active and inactive X chromosomes. This suggests the potential use of somatic-cell hybrids containing either a maternal or a paternal human chromosome as a model system to study known imprinted genes and to identify as-yet-unknown imprinted genes. Testing gene expression by using reverse transcription followed by PCR, we show that functional imprints are maintained at four previously characterized 15q11–q13 loci in hybrids containing a single human chromosome 15 and at two chromosome 11p15 loci in hybrids containing a single chromosome 11. In contrast, three γ-aminobutyric acid type A receptor subunit genes in 15q12–q13 are nonimprinted. Furthermore, we have found that differential DNA methylation imprints at the SNRPN promoter and at a CpG island in 11p15 are also maintained in somatic-cell hybrids. Somatic-cell hybrids therefore are a valid and powerful system for studying known imprinted genes as well as for rapidly identifying new imprinted genes.