Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes.

In this study, we assess the evolutionary relationships among different chromosomal copies of a subtelomeric block of sequence. This block contains homology to three olfactory receptor genes and is dispersed on at least 14 different chromosome ends in humans. It is single-copy in non-human primates. We analyzed single nucleotide polymorphisms in two 1 kb subregions and a polymorphic Alu insertion within 181 copies of this block from 12 chromosome ends and found evidence for recent interactions between the subtelomeric regions of non-homologous chromosomes. First, several sequence haplotypes are each present on multiple chromosomes, and several chromosomes each have multiple alleles with divergent haplotypes. Secondly, the observed variation clearly indicates that chromosomes 5q, 8p, 11p and/or 15q have each received the block from at least two different sources by non-homologous exchange. In addition, we observe at least one ectopic gene conversion event. Awareness of such exchange among sequences on non-homologous chromosomes is critical for accurate analysis of these complex and dynamic regions of the genome.

Transcriptional activity of multiple copies of a subtelomerically located olfactory receptor gene that is polymorphic in number and location.

We report here on the transcriptional activity of multiple copies of a subtelomerically located olfactory receptor (OR) gene, OR-A. Due to recent duplication events, both the copy number and chromosomal location of OR-A vary among humans. Sequence analyses of 180 copies of this gene, derived from 12 chromosome ends in 22 individuals, show that the main coding exon of all but one copy is an intact open reading frame with 0-5 predicted amino acid differences. We detected transcription of OR-A in both olfactory epithelium and testis tissue using RT-PCR amplification with primers designed on the basis of a computationally predicted gene structure. Two alternatively spliced forms of transcripts, one encoding an isoform with an extended N-terminus, were found in both tissues. A third transcript, derived from a second promoter, was also observed in testes. The start methionine is predicted in all transcripts to lie in an upstream exon rather than the main coding exon, as is typical for most other OR genes. By examining sequence variants among transcripts, we show that transcription of this gene occurs at multiple chromosomal locations. Our results lend credence to the idea that OR diversity could be generated in rearrangement-prone subtelomeric regions and show that polymorphism in subtelomeric regions could lead to individual-to-individual variation in the expressed repertoire of OR genes.

Modeling the feasibility of whole genome shotgun sequencing using a pairwise end strategy.

In pairwise end sequencing, sequences are determined from both ends of random subclones derived from a DNA target. Sufficiently similar overlapping end sequences are identified and grouped into contigs. When a clone's paired end sequences fall in different contigs, the contigs are connected together to form scaffolds. Increasingly, the goals of pairwise strategies are large and highly repetitive genomic targets. Here, we consider large-scale pairwise strategies that employ mixtures of subclone sizes. We explore the properties of scaffold formation within a hybrid theory/simulation mathematical model of a genomic target that contains many repeat families. Using this model, we evaluate problems that may arise, such as falsely linked end sequences (due either to random matches or to homologous repeats) and scaffolds that terminate without extending the full length of the target. We illustrate our model with an exploration of a strategy for sequencing the human genome. Our results show that, for a strategy that generates 10-fold sequence coverage derived from the ends of clones ranging in length from 2 to 150 kb, using an appropriate rule for detecting overlaps, we expect few false links while obtaining a single scaffold extending the length of each chromosome.

Changes in cell surface molecules associated with in vitro culture of prostatic stromal cells.

BACKGROUND: Prostate stromal cells can be readily cultured in vitro. Are these proliferating cells representative of stromal cells in situ? Since the expression of cell surface molecules, like the cluster of differentiation (CD) antigens, can be affected by changes in physiological conditions cultured stromal cells may differ from uncultured stromal cells in their complement of CD molecules. METHODS: Prostate stromal cells were prepared from tissue specimens and cultured. Flow cytometry was used to analyze the expression of 107 CD molecules in the resultant cells. Expression of the CD molecules by prostate cells in situ was done by immunohistochemistry. RESULTS: The expression of a number of cell surface molecules such as CD10, CD13, CD26, and CD44 is elevated in prostatic stromal cells cultured in vitro. These are markers of epithelial cells in tissue. Other molecules expressed by the cultured stromal cells include CD29, CD49a, CD49b, CD49d, CD49f, CD51/61, CD54, CD55, CD56, CD58, CD59, CD61, CD71, CD79b, CD81, CD82, CD90, CD95, CD107a, CD130, and CD147. Among these are stromal, epithelial, and nonstromal/nonepithelial markers as defined by tissue immunohistochemistry. CONCLUSION: Cultured stromal cells express a number of CD molecules normally found in other cell types of the prostate. Cells can express different CD molecules under different conditions.

Polarization of scatter and fluorescence signals in flow cytometry.

BACKGROUND: The pulses of light scatter and fluorescence measured in flow cytometers exhibit varying degrees of polarization. Flow cytometers are heterogeneously sensitive to this polarization, depending on the light source(s), the optical layout, and the types of mirrors and filters used. Therefore, fluorescence polarization can affect apparent intensity ratios between particles and interfere with schemes for interlaboratory standardization. METHODS: We investigate the degree to which polarization affects common flow cytometry measurements. Our technique for determining polarization differs from previous methods because complete distributions of intensity versus polarization angle are measured, rather than intensities at just two orthogonal polarization angles. Theoretical models for scatter and fluorescence are presented and verified by making polarization measurements of calibration beads. RESULTS: Measurements of cells stained with a variety of dyes illustrate that fluorescence polarization occurs frequently in flow cytometry. CONCLUSIONS: Consequences for quantitative cytometry are discussed, and the use of the "magic angle" to make a flow cytometer insensitive to fluorescence polarization is proposed.

Analysis and sorting of prostate cancer cell types by flow cytometry.

BACKGROUND: Prostate tumor heterogeneity as manifested by differential expression of markers can be attributed to multiple types of cancer cells populating a tumor. Does the composition differ between primary tumor and metastasis? How can one isolate the different cancer cell types to study? What is the relationship among cancer cell types? METHODS: Flow cytometry keying on the prostate epithelial cell surface markers CD57 and CD44 was applied to analyze and sort single cells prepared from tumor tissue samples by collagenase digestion. In normal tissue, CD57 is found on luminal cells and CD44 on basal cells. RESULTS: CD57(+) and CD44(+) cells were sorted from various prostate tumor tissue specimens. The CD57(+) cancer cell type was found to predominate in primary tumors, while the CD44(+) cancer cell type was found to predominate in two visceral metastases. All tumors could be characterized by a ratio of CD57(+) and CD44(+) cancer cells. CONCLUSIONS: Two types of prostate cancer cells, CD57(+) and CD44(+), were identified. The finding that most primary tumors contain a predominantly CD57(+) cancer cell population agrees with the argument that cancer cells arise from the transformation of CD57(+) luminal cells. However, CD44(+) cancer cells are also present in some primary tumors; and in some metastases, they, and not CD57(+) cells, constitute a predominant population.

Sharp DNA bends as landmarks of protein-binding sites on straightened DNA.

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.

Characterization of differentially expressed genes in purified Drosophila follicle cells: toward a general strategy for cell type-specific developmental analysis.

Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. We describe a method for identifying genes expressed in the follicle cells with potential roles in axis formation. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from our pilot screens include (i) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers, (ii) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (iii) the identification of a Drosophila homologue of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers.

Analysis of sequence-tagged-connector strategies for DNA sequencing.

The BAC-end sequencing, or sequence-tagged-connector (STC), approach to genome sequencing involves sequencing the ends of BAC inserts to scatter sequence tags (STCs) randomly across the genome. Once any BAC or other large segment of DNA is sequenced to completion by conventional shotgun approaches, these STC tags can be used to identify a minimum tiling path of BAC clones overlapping the nucleation sequence for sequence extension. Here, we explore the properties of STC-sequencing strategies within a mathematical model of a random target with homologous repeats and imperfect sequencing technology to understand the consequences of varying various parameters on the incidence of problem clones and the cost of the sequencing project. Problem clones are defined as clones for which either (A) there is no identifiable overlapping STC to extend the sequence in a particular direction or (B) the identified STC with minimum overlap comes from a nonoverlapping clone, either owing to random false matches or repeat-family homology. Based on the minimum overlap, we estimate the number of clones to be entirely sequenced and, then, using cost estimates, identify the decision rule (the degree of sequence similarity required before a match is declared between an STC and a clone) to minimize overall sequencing cost. A method to optimize the overlap decision rule is highly desirable, because both the total cost and the number of problem clones are shown to be highly sensitive to this choice. For a target of 3 Gb containing approximately 800 Mb of repeats with 85%-90% identity, we expect <10 problem clones with 15 times coverage by 150-kb clones. We derive the optimal redundancy and insert sizes of clone libraries for sequencing genomes of various sizes, from microbial to human. We estimate that establishing the resource of STCs as a means of identifying minimally overlapping clones represents only 1%-3% of the total cost of sequencing the human genome, and, up to a point of diminishing returns, a larger STC resource is associated with a smaller total sequencing cost.

Spin-stretching of DNA and protein molecules for detection by fluorescence and atomic force microscopy.

We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl2-soaked mica surface. We present fluorescent images of straightened lambdaDNA molecules and AFM images of stress-shared, reduced von Willebrand factor as well as straightened lambdaDNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.

Mapping a protein-binding site on straightened DNA by atomic force microscopy.

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface. The immobilized molecules were detected by AFM. The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site. Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site. The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.

Large multi-chromosomal duplications encompass many members of the olfactory receptor gene family in the human genome.

The human genome contains thousands of genes that encode a diverse repertoire of odorant receptors (ORs). We report here on the identification and chromosomal localization of 74 OR-containing genomic clones. Using fluorescence in situ hybridization (FISH), we demonstrate a striking homology among a set of approximately 20 OR locations, illustrating a history of duplications that have distributed OR sequences across the genome. Half of the OR-containing BACs cloned from total genomic DNA and 86% of cosmids derived from chromosome 3 cross-hybridize to a subset of these locations, many to 17 of them. These paralogous regions are distributed on 13 chromosomes, and eight lie in terminal bands. By analyzing clones from an approximately 250 kb clone-walk across one of these sites (3p13), we show that the homology among these sites is extensive (>150 kb) and encompasses both OR genes and intergenic genomic sequences. The FISH signals appear significantly larger at some sites than at the native location, indicating that portions of some duplicons have undergone local amplification/attrition. More restricted duplications involving pairs of other genomic locations are detected with 12% of the OR-BACs. Only a small subset of OR locations is sufficiently diverged from the others that clones derived from them behave as single-copy FISH probes. We estimate that duplications encompassing members of the OR gene family account for >0.1% of the human genome. A comparison of FISH signals at orthologous locations in other primates indicates that a portion of this OR 'subgenome' has been in flux during the divergence of primates, possibly as a mechanism for evolving the repertoire of olfactory receptors.

A gene recently inactivated in human defines a new olfactory receptor family in mammals.

The olfactory receptor (OR) gene family constitutes one of the largest multigene families and is distributed among many chromosomal sites in the human genome. Four OR families have been defined in mammals. We previously demonstrated that a high fraction of human OR sequences have incurred deleterious mutations, thus reducing the repertoire of functional OR genes. In this study, we have characterized a new OR gene, 912-93, in primates. This gene is unique and it defines a new OR family. It localizes to human chromosome 11q11-12 and at syntenical sites in other hominoids. The sequence marks a previously unrecognized rearrangement of pericentromeric material from chromosome 11 to the centromeric region of gibbon chromosome 5. The human gene contains a nonsense point mutation in the region corresponding to the extracellular N-terminus of the receptor. This mutation is present in humans of various ethnic groups, but is absent in apes, suggesting that it probably appeared during the divergence of humans from other apes, <4 000 000-5 000 000 years ago. A second mutation, a frameshift at a different location, has occurred in the gorilla copy of this gene. These observations suggest that OR 912-93 has been recently silenced in human and gorilla, adding to a pool of OR pseudogenes whose growth may parallel a reduction in the sense of smell in primates.

Optimization of restriction fragment DNA mapping.

Consider a mapping project in which overlap of clonal segments is inferred from complete multiple restriction digests. The fragment sizes of the clones are measured with some error, potentially leading to a map with erroneous links. The number of errors in the map depends on the number and types of enzymes used to characterize the clones. The most critical parameter is the decision rule k, or the criterion for declaring clone overlap. Small changes in k may cause an order of magnitude change in the amount of work it takes to build a map of given completion. We observe that the cost of an optimal mapping strategy is approximately proportional to the target size. While this finding is encouraging, considerable effort is nonetheless required: for large-scale sequencing projects with up-front mapping, mapping will be a non-negligible fraction of the total sequencing cost.

Expectation and variance of true and false fragment matches in DNA restriction mapping.

Consider a DNA mapping project in which overlap of clones is inferred from multiple complete restriction enzyme digests. Each enzyme cuts each clone randomly into fragments whose lengths are determined with some error. Clones that share fragments with matching lengths could contain a region of overlap. However, common fragment lengths may be due to random coincidence leading to a false overlap declaration. Although the probability of false fragment matching is small, a mapping project involves a large number of clone comparisons. Consequently, erroneous fragment matches can be a serious problem. We use a geometrical probability approach to develop exact integral formulas and first-order approximations for the expected number and variance of classes of fragment pairs that will be identified falsely as matching. We also find exact formulas for the expected value, and variance of the number of true fragment matches. These formulas are useful in comparing different mapping strategies.

Trapping of DNA in nonuniform oscillating electric fields.

DNA molecules can be manipulated in aqueous solution in a manner analogous to optical trapping. Due to the induction of an electric dipole, DNA molecules are pulled by a gradient force to regions of high electric field strength. Molecules can be locally trapped in an oscillating field using strips of very thin gold film to generate strong electric fields with steep gradients. Spatial control over the trapped molecules is achieved because they are confined to a width of approximately 5 microm along the edges of the gold-film strips. By mixing static and oscillating electric fields, trapped molecules can be moved from one edge to another or made to follow precise trajectories along the edges. This phenomenon should be useful in microdevices for manipulation of small quantities or single molecules of DNA.

Distribution of olfactory receptor genes in the human genome.

We demonstrate that members of the olfactory receptor (OR) gene family are distributed on all but a few human chromosomes. Through FISH analysis, we show that OR sequences reside at more than 25 locations in the human genome. Their distribution is biased for terminal bands. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence-relationships are indicative of the inter- and intrachromosomal duplications responsible for OR family expansion. The human genome has accumulated a striking number of dysfunctional copies: 72% of the sequences are pseudogenes. ORF-containing sequences predominate on chromosomes 7, 16 and 17.

Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes.

We have identified three new members of the olfactory receptor (OR) gene family within a large segment of DNA that is duplicated with high similarity near many human telomeres. This segment is present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. Additional copies are present polymorphically at 11 other subtelomeric locations. The frequency with which the block is present at some locations varies among populations. While humans carry seven to 11 copies of the OR-containing block, it is located in chimpanzee and gorilla predominantly at a single site, which is not orthologous to any of the locations in the human genome. The observation that sequences flanking the OR-containing segment are duplicated on larger and different sets of chromosomes than the OR block itself demonstrates that the segment is part of a much larger, complex patchwork of subtelomeric duplications. The population analyses and structural results suggest the types of processes that have shaped these regions during evolution. From its sequence, one of the OR genes in this duplicated block appears to be potentially functional. Our findings raise the possibility that functional diversity in the OR family is generated in part through duplications and inter-chromosomal rearrangements of the DNA near human telomeres.