Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

A plant-transformation-competent BIBAC library from the Arabidopsis thaliana Landsberg ecotype for functional and comparative genomics.

The genome of the model plant Arabidopsis thaliana has been sequenced to near completion. To facilitate experimental determination of the function of every gene in the species, we constructed a large-insert library from the Landsberg ecotype using a plant-transformation-competent binary BAC vector, BIBAC2. The library contains 11,520 clones with an estimated average insert size of 162 kb. Of a sample of 102 clones, 17.6% had no inserts; further, in the library as a whole, 287 clones contained chloroplast DNA, and 25 contained mitochondrial DNA. Thus it is estimated that 9,295 clones originated from the nuclear genome, representing a 11.5 x coverage. The library was further characterized by screening with probes corresponding to 180-bp repeats, 5S rDNA, 18S-25S rDNA and 23 single-copy RFLP markers. The results showed that 92 clones contained 180-bp centromeric repeats, 78 contained 5S rDNA and 95 contained 18S-25S rDNA, approximately 1%, 0.8% and 1%, respectively, of the nuclear clones in the library. Screening the library with the 23 RFLP markers showed that each one hybridized to an average of seven clones. This library is the first large-insert DNA library for the widely studied Landsberg erecta strain. It will greatly facilitate gene identification by complementation screening, and will enhance analysis of the structure, organization and evolution of the A. thaliana genome.

Crossover interference in Arabidopsis.

The crossover distribution in meiotic tetrads of Arabidopsis thaliana differs from those previously described for Drosophila and Neurospora. Whereas a chi-square distribution with an even number of degrees of freedom provides a good fit for the latter organisms, the fit for Arabidopsis was substantially improved by assuming an additional set of crossovers sprinkled, at random, among those distributed as per chi square. This result is compatible with the view that Arabidopsis has two pathways for meiotic crossing over, only one of which is subject to interference. The results further suggest that Arabidopsis meiosis has >10 times as many double-strand breaks as crossovers.

Tetrad analysis in higher plants. A budding technology.

Tetrad analysis, the ability to manipulate and individually study the four products of a single meiotic event, has been critical to understanding the mechanisms of heredity. The Arabidopsis quartet (qrt) mutation, which causes the four products of male meiosis to remain attached, enables plant biologists to apply this powerful tool to investigations of gamete development, cell division, chromosome dynamics, and recombination. Here we highlight several examples of how qrt has been used to perform tetrad analysis and suggest additional applications including a genetic screen for gametophytic mutants and methods for investigating gene interactions by synthetic lethal analysis.

Centromeres in the genomic era: unraveling paradoxes.

The centromeres of higher plants and animals share many common features, though current models fail to account for all aspects of centromere composition and function. This dilemma is likely to be resolved in the next few years in Arabidopsis where robust assays for centromere function are available and the sequence of the entire genome will be determined.

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Genetic definition and sequence analysis of Arabidopsis centromeres.

High-precision genetic mapping was used to define the regions that contain centromere functions on each natural chromosome in Arabidopsis thaliana. These regions exhibited dramatic recombinational repression and contained complex DNA surrounding large arrays of 180-base pair repeats. Unexpectedly, the DNA within the centromeres was not merely structural but also encoded several expressed genes. The regions flanking the centromeres were densely populated by repetitive elements yet experienced normal levels of recombination. The genetically defined centromeres were well conserved among Arabidopsis ecotypes but displayed limited sequence homology between different chromosomes, excluding repetitive DNA. This investigation provides a platform for dissecting the role of individual sequences in centromeres in higher eukaryotes.

Assaying genome-wide recombination and centromere functions with Arabidopsis tetrads.

During meiosis, crossover events generate new allelic combinations, yet the abundance of these genetic exchanges in individual cells has not been measured previously on a genomic level. To perform a genome-wide analysis of recombination, we monitored the assortment of genetic markers in meiotic tetrads from Arabidopsis. By determining the number and distribution of crossovers in individual meiotic cells, we demonstrated (i) surprisingly precise regulation of crossover number in each meiosis, (ii) considerably reduced recombination along chromosomes carrying ribosomal DNA arrays, and (iii) an inversely proportional relationship between recombination frequencies and chromosome size. This use of tetrad analysis also achieved precise mapping of all five Arabidopsis centromeres, localizing centromere functions in the intact chromosomes of a higher eukaryote.

RFLP and physical mapping with an rDNA-specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4.

Ribosomal RNA genes are organized in tandem arrays called nucleolus organizer regions (NORs). In a prior study, RFLP mapping on pulsed-field gels placed NOR2 at the northern tip of Arabidopsis thaliana chromosome 2. New polymorphisms have allowed the other NOR, NOR4, to be mapped to the northern tip of chromosome 4. To map NOR-associated loci, rDNA-specific cleavage by I-Ppol, an endonuclease with a 15 nucleotide recognition sequence involved in rDNA-homing of a mobile, self-splicing Group I intron in Physarum was exploited. I-Ppol digestion of A. thaliana genomic DNA liberated two telomere-containing fragments no larger than 13 kbp, and telomere polymorphisms identified using I-Ppol cosegregated with NOR2 and NOR4. Restriction mapping suggested that telomere-proximal rRNA genes are oriented with their 5' ends nearest the chromosome ends and their 3' ends nearest the centromere. This orientation was confirmed using the polymerase chain reaction to clone one of the telomere-rDNA junctions, most likely the junction on chromosome 4. The telomeric repeats join the terminal rRNA gene downstream of its promoter, suggesting that this first gene is inactive. Subtelomeric repetitive DNAs are absent at the telomere-rDNA junction. Localization of NOR2, NOR4 and their associated telomeres, TEL2N and TEL4N, respectively, provides end points for the genetic and physical maps of chromosomes 2 and 4.

Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution.

Eukaryotic genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNA (rRNA genes or rDNA) are virtually identical within a species, yet they evolve rapidly between species, a phenomenon known as concerted evolution. The mechanisms by which sequence homogenization and fixation of new rRNA gene variants occurs within a genome are not clear. In diploid Arabidopsis thaliana, approximately 1500 rRNA genes are tandemly arrayed at two nucleolus organizer regions, one on chromosome 2 (NOR2), the other on chromosome 4 (NOR4). This paper shows that NOR2 and NOR4 are similar in size, each spanning approximately 3.5-4.0 Mbp. Using two-dimensional mapping techniques involving a combination of pulsed-field and conventional gel electrophoresis, the distributions of four distinct rRNA gene variants at NOR2 and NOR4 have been determined. rRNA genes at NOR4 are homogeneous with respect to a HindIII site occurring once per gene. In contrast, fewer than 10% of the rRNA genes at NOR2 are HindIII-bearing variants. A single intergenic spacer length is found among rRNA genes at NOR2 but three classes of spacer length variants are present at NOR4. The NOR4 variants are not intermingled with one another; instead, they are highly clustered over distances as large as 1.5 Mbp. These data suggest that in the concerted evolution of rRNA genes, homogenization is a consequence of local spreading of new rRNA gene variants.

Epicardial surface dynamics in the closed-chest normal canine.

Past studies of the changing three-dimensional shape of the heart in the closed chest during the cardiac cycle have been restricted to the measurement of local deformations at a relatively few specific locations, and often have required surgical procedures that alter the measurements obtained. In the study reported here, high precision displacement and velocity measurements were obtained at the epicardial interface using a Compton backscatter imaging technique that does not require a surgical intervention or contrast injections. Displacement and velocity measurements were obtained at more than 200 locations at the epicardial interface at 13 ms intervals throughout the cardiac cycle. Measurements of the changing shape of the heart during the cardiac cycle with this technique are precise to 0.1 mm (S.D.). Displacement and velocity patterns recorded in this study confirm and integrate the studies of many others and also add new information. An unexpected vigorous inward motion of both the LV (39 mm s-1) and RV (26 mm s-1) surfaces during isovolumic relaxation and early rapid refill is demonstrated. Velocities during this period equal or exceed those that occur during ejection. During ejection, inward LV motion at the base of the heart precedes that at the apex by 80-90 ms. Posterior LV displacements and velocities during ejection are 4-6 times greater than those at the anterior and apex. The Compton backscatter imaging technique for obtaining undisturbed measurements of cardiac dynamics in the closed chest has potential as a non-invasive clinical tool for serial studies of cardiac surface motion abnormalities. The data presented can also be used to set surface boundary conditions for biomechanical models of heart deformation.

Use of RFLPs larger than 100 kbp to map the position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana.

In Arabidopsis thaliana ribosomal RNA genes (rRNA genes or rDNA) are grouped in two nucleolus organizer regions(NORs) that together comprise approximately 6% of the genome. The map positions of the NORs relative to other genetic markers are unknown. It was found that the restriction endonuclease Hindlll cuts once in some but not all rRNA genes to yield strain-specific RFLPs of 100-700 kb that could be visualized by pulsed-field gel electrophoresis and Southern blotting. The Hindlll RFLPs of the A. thaliana strains Columbia and Landsberg segregated among recombinant inbred lines derived from a cross between these two strains. Linkage analysis placed the NOR bearing the polymorphic Hindlll sites to the top of the upper arm of chromosome 2. The name NOR2 is suggested for this locus. Hindlll-bearing rRNA genes ere interspersed with clusters of Hindlll-less genes throughout NOR2. The observed clustering is most consistent with unequal crossing-over, or nearest-neighbor gene conversion, as the mechanism(s) that spread rRNA gene variants throughout an NOR. No meiotic cross-over events yielding a 'hybrid' NOR with multiple RFLPs from both parents were observed among the 47 recombinant inbred lines examined. However,the appearance of novel Hindlll profiles in approximately 40% of the recombinant inbred lines demonstrates that fluctuations in the distribution of rRNA gene variants occur frequently and can be readily detected on pulsed-field gels.

The RNA polymerase I transactivator upstream binding factor requires its dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA.

Upstream binding factor (UBF) is an important transactivator of RNA polymerase I and is a member of a family of proteins that contain nucleic acid binding domains named high-mobility-group (HMG) boxes because of their similarity to HMG chromosomal proteins. UBF is a highly sequence-tolerant DNA-binding protein for which no binding consensus sequence has been identified. Therefore, it has been suggested that UBF may recognize preformed structural features of DNA, a hypothesis supported by UBF's ability to bind synthetic DNA cruciforms, four-way junctions, and even tRNA. We show here that full-length UBF can also bend linear DNA to mediate circularization of probes as small as 102 bp in the presence of DNA ligase. Longer probes in the presence of UBF become positively supercoiled when ligated, suggesting that UBF wraps the DNA in a right-handed direction, opposite the direction of DNA wrapping around a nucleosome. The dimerization domain and HMG box 1 are necessary and sufficient to circularize short probes and supercoil longer probes in the presence of DNA ligase. UBF's sequence tolerance coupled with its ability to bend and wrap DNA makes UBF an unusual eukaryotic transcription factor. However, UBF's ability to bend DNA might explain how upstream and downstream rRNA gene promoter domains interact. UBF-induced DNA wrapping could also be a mechanism by which UBF counteracts histone-mediated gene repression.

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids.

Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences.

Inward diastolic motion: a normal component of isovolumic relaxation and rapid refill.

Many reports note expansive events occurring in the left ventricle during isovolumic relaxation. Expansions during isovolumic relaxation require compensatory inward displacements elsewhere in the ventricle. The location and character of such compensatory displacements have been a continuing source of speculation. Using a high-precision Compton backscatter imaging (CBI) technique, we have detected an early diastolic inward motion that initiates during isovolumic relaxation on the right and left epicardial free walls of the heart in 100% of the 14 normal canines we have studied. This inward motion is first detected 20-30 ms after left ventricular maximal rate of pressure decrease over time (-dP/dtmax), lasts into the early rapid filling phase with a mean duration of 92 +/- 5 (SE) ms, and ends approximately 30 ms after opening of the mitral valve. Maximum wall velocities during this time period (approximately 20 mm/s) exceed those occurring in the same regions during systole. Inward surface displacements in the areas undergoing inward motion average 1.1 +/- 0.2 and 0.9 +/- 0.2 mm on the left and right side of the heart, respectively.

The effects of pacing site on left ventricular epicardial surface velocity patterns during systole.

Changes in epicardial LV velocity patterns during isovolumic contraction and ejection as induced by ventricular pacing were studied in 15 canines. A noninvasive imaging technique that provided high temporal resolution was used to study the timing of an outward expansion of the LV during isovolumic contraction and the propagation pattern of an inward LV velocity wavefront during ejection. With this technique, surface displacements were measured (+/- 0.1 mm SD) at 50-70 locations on the LV free wall at 5-msec intervals. Velocities were calculated by differentiating the surface displacement waveforms and an interpolation procedure was used to provide detailed color coded velocity maps of the LV surface. LV surface velocities were determined from data obtained during closed-chest endocardial pacing from each of four sites: right atrium, right ventricular apex, left ventricular apex, and right ventricular outflow tract. These surface velocities showed a distinct spatial and temporal pattern for each pacing site. The results show that this noninvasive mapping procedure has potential for determining the location of an ectopic ventricular focus.

Analysis of edge-tracking errors inherent in fluoroscopic images of the beating heart.

RATIONALE AND OBJECTIVES: Because of the complex relationships between the dynamic three-dimensional cardiac surface shape and its projected image, errors arise with the use of two-dimensional silhouettes to measure displacements of the heart. The character and frequency of such errors are examined. METHODS: A high-precision x-ray scatter imaging technique was used to reconstruct the three-dimensional shape of the left ventricular free wall throughout the cardiac cycle. Displacements of the three-dimensional surface were then compared with those on the two-dimensional projected silhouette. Silhouette displacement errors were determined as a function of time during the cardiac cycle and variability between hearts. RESULTS: Differences between silhouette measurements and those on the cardiac surface range from 0% to 125% of peak-to-peak displacements occur, along 33% to 75% of the silhouette contours and cover 66% of the cardiac cycle. CONCLUSION: Two-dimensional silhouette displacements provide inconsistent measurements of motion patterns on the three-dimensional cardiac surface.

A flying spot X-ray system for Compton backscatter imaging.

A Compton X-ray backscatter imaging (CBI) system using a single detector and a mechanically rastered "flying spot" X-ray beam has been designed, built, and tested. While retaining the essential noninvasive imaging capability of previous multiple detector CBI devices, this single detector system incorporates several advances over earlier CBI devices: more efficient detection of scattered X-rays, reduced X-ray exposure, and a simplified scan protocol more suitable for use with humans. This new CBI system also has specific design features to permit automating data acquisition from multiple two-dimensional image planes for integration into a 3D dynamic surface image. A simulated multislice scan study of a human thorax phantom provided X-ray dosimetry data verifying a very low X-ray dose (~50 mrem) delivered by this imaging device. Validation experiments with mechanical models show that surface displacement at typical heart beat frequencies can be measured to the nearest 0.1 mm (SD).

Ischemia-induced depressed systolic thickening is transiently augmented by remote coronary occlusion.

To determine if ischemia-induced depressed myocardial thickening can be augmented by remote coronary occlusion, posterior wall function (pulsed Doppler crystal) was measured before and after left anterior descending coronary artery occlusion in the presence of reduced circumflex coronary artery flow (of sufficient severity to reduce resting function) in an anesthetized open-chest canine preparation in which the circumflex was pump-perfused with carotid arterial blood. Left anterior descending coronary occlusion elicited an immediate significant increase in posterior bed thickening fraction (TF%) (3.7 +/- 1.5 to 5.9 +/- 1.3%), but by 135 sec TF% had again deteriorated. The transient increase in thickening was not caused by increased flow to the posterior bed (microspheres, n = 3), nor was it related to a Frank-Starling mechanism (echocardiography, n = 3). Despite an ischemic-induced reduction in systolic shortening, systolic thickening can be transiently augmented by remote coronary occlusion. The etiology may be related to systolic unloading.

Systolic thickening increases from subepicardium to subendocardium.

STUDY OBJECTIVE: The aim was to determine the relation between systolic function and depth below the epicardial surface, using pulsed Doppler crystals. DESIGN: Systolic excursion in mm was measured at multiple depths below the epicardial surface. Measurements were made during control conditions. SUBJECTS: Thirty four anaesthetised mongrel dogs were studied. MEASUREMENTS AND MAIN RESULTS: A total of 333 measurements of systolic excursion were made during control conditions at 1 to 3 crystal sites per dog. Two models were constructed; the model which assumed a quadratic relation between depth (d) and systolic excursion (EX) (EX = 0.498 + 0.176d + 0.006d2) fit the data better than did a model which assumed a linear relation (EX = -0.0716 + 0.254d). CONCLUSIONS: These data support the prediction from geometric models that systolic excursion increases in a non-linear fashion from subepicardium to subendocardium.