Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia.

DNA rearrangements caused by chromosome translocations between band 11q23 and various chromosomes can be detected by a single probe, B859, an 859-base pair complementary DNA fragment derived from the human ALL-1 gene. To try to understand why band 11q23 becomes a frequent target of the translocations, we have sequenced the entire breakpoint cluster region, a 8342-base pair BamHI genomic fragment delineated by B859. We found eight Alu repeats located within this region in the same orientation as the ALL-1 gene. We have also analyzed the sequences of the breakpoints in 10 patients with 6 different types of 11q23 aberration. In five patients the breaks coincided with Alu sequences on chromosome 11, but not on the partner chromosomes. Also, seven of the breaks occurred in the region delineated by exons 6 and 7, which is composed mainly of Alu sequences. In three patients topoisomerase II recognition site-like sequences, at different stringency levels, were identified at the breakpoints on chromosome 11. We conclude that while there is no specific sequence element present at all the breakpoints, the high density of Alu sequences in the breakpoint cluster region possibly makes the latter more prone to recombination events.

Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation.

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;11) translocation predominating among acute lymphoblastic leukemias, and the t(9;11), t(11;19) and t(6;11) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and constituting the human homologue of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. Here we report the cloning and the characterization of the partner gene from chromosome 6 (AF-6). AF-6 is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduction at special cell-cell junctions.

Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs.

Chromosome translocations involving band 11q23 are associated with human acute leukemias. These translocations fuse the ALL-1 gene, homolog of Drosophila trithorax and located at chromosome band 11q23, to genes from a variety of chromosomes. We cloned and sequenced cDNAs derived from transcripts of the AF-4 and AF-9 genes involved in the most common chromosome abnormalities, t(4:11)(q21:q23) and t(9:11)(p22:q23), respectively. Sequence analysis indicates high homology between the AF-9 gene protein product and the protein encoded by the ENL gene fused to ALL-1 in (11:19) chromosome translocations. AF-4, AF-9, and ENL proteins contain nuclear targeting sequences as well as serine-rich and proline-rich regions. Stretches abundant in basic amino acids are also present in the three proteins. These results suggest that the different proteins fused to ALL-1 polypeptide(s) provide similar functional domains.

Characterization of drought resistance in a wild relative of wheat, Triticum kotschyi.

Wild relatives of wheat have served as a genetic source for economically useful traits. A better understanding of the mechanisms underlying such traits may be useful in the genetic transfer and selection processes. Research was undertaken to compare the effects of controlled water stress on photosynthetic parameters in Triticum kotschyi, a drought resistant wild wheat and Triticum aestivum cv. Lakhish, a drought sensitive wheat cultivar. During stress development, the leaf water potential decreased at a slower rate, and the quantum yield of oxygen evolution, measured photoacoustically in vivo, decreased to a smaller extent in the drought resistant wild wheat than in the wheat cultivar. The decrease in quantum yield at water potentials from -0.9 Mpa down to -2.3 Mpa was not accompanied by damage to PS II reaction centers as there was no change in variable fluorescence. Below -2.3 Mpa the fluorescence yield of both species decreased indicating loss of intrinsic efficiency of PS II. The osmotic potential of cell sap was found to decrease at the same rate in both species at high hydration states. Proline accumulated to a much greater extent in the wild wheat as compared to the cultivated wheat as a result of water stress. Drought resistance was also examined in relation to thylakoid membrane fluidity measured by fluorescence polarization. Thylakoid membrane fluidity was fully maintained in the wild wheat, but decreased substantially in the wheat cultivar, at equal tissue water potentials below -1.9 Mpa. One mechanism for maintaining the higher quantum yield of oxygen evolution during severe stress (at water potentials below -1.9 Mpa), may involve the greater stability of thylakoid membrane fluidity in the wild wheat.

The (4;11)(q21;q23) chromosome translocations in acute leukemias involve the VDJ recombinase.

Chromosomal region 11q23 is frequently rearranged in acute lymphocytic leukemias (ALLs) and in acute myeloid leukemias (AMLs), mostly in reciprocal exchanges with various translocation partners. The most common of these translocations is t(4;11)(q21;q23). It is present in approximately 10% of ALL patients, most frequently in very young children. We have recently cloned a region of chromosome 11, the ALL-1 locus, found to be rearranged in malignant cells from patients with the t(4;11), t(9;11), t(11;19), t(1;11), t(6;11), t(10;11), and del(11q23) chromosomal abnormalities. Here we report the cloning and characterization of chromosomal breakpoints from leukemic cells with t(4;11) aberrations. The breakpoints cluster in regions of 7-8 kilobases on both chromosomes 4 and 11. The presence of heptamer- and nonamer-like sequences at the sites of breakage suggests that the VDJ recombinase utilized for immunoglobulin gene rearrangement is also directly involved in these translocations. We also show that leukemic cells with t(4;11) express altered RNAs transcribed from the derivative chromosomes 11 and 4.

The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene.

The ALL-1 gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 4, 6, 9, 10, or 19. The gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of more than 3910 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene, including cysteine-rich regions that can be folded into six zinc finger-like domains. The breakpoint cluster region within ALL-1 spans 8 kb and encompasses several small exons, most of which begin in the same phase of the open reading frame. The t(4;11) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-1 and from a gene on chromosome 4. This suggests that each 11q23 abnormality gives rise to a specific oncogenic fusion protein.

An altered 11-kilobase transcript in leukemic cell lines with the t(4;11)(q21;q23) chromosome translocation.

The 11q23 chromosome band is frequently associated with chromosomal aberrations in human leukemias. We have previously cloned a DNA fragment derived from chromosome 11 which could be used as a probe to detect rearrangements in DNAs from the leukemic cells of patients with the t(4;11), t(9;11), and t(11;19) translocations. In this study we now show that the same probe detects DNA rearrangements in malignant cells from patients with the t(1;11), t(6;11), t(10;11), and del (11q23) chromosomal abnormalities. A second probe obtained from a region located centrometric to the breakpoint cluster detects major and minor transcripts of 12.5 and 11.5 kilobases, respectively, in all cell lines examined. The same probe identifies an altered 11-kilobase RNA in all three independent cell lines with the t(4;11)(q21;q23) chromosome translocation.

Cloning of ALL-1, the locus involved in leukemias with the t(4;11)(q21;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13) chromosome translocations.

Chromosomal region 11q23 participates in a number of reciprocal translocations with specific regions of chromosomes 4, 9, 19, and others. These translocations are associated with acute lymphocytic leukemia and acute myelomonocytic, monocytic, and myelogenous leukemia. From a yeast artificial chromosome containing human DNA derived from 11q23 we cloned a DNA fragment which can be used as a probe to detect rearrangements in leukemic cells from the majority of patients with the t(4;11), t(9;11), and t(11;19) translocations. The breakpoints cluster in a small DNA region of less than 5.8 kilobases.

The use of photothermal radiometry in assessing leaf photosynthesis: I. General properties and correlation of energy storage to P700 redox state.

Energy storage measurements by modulated photothermal radiometry (PTR) were carried out on intact leaves to assess the value of the PTR method for photosynthesis research. In particular, correlations to the redox state of P700 under various conditions were examined. PTR monitors modulated light conversion to heat by sensing the resulting modulated infra-red radiation emitted from the leaf. It is, therefore, a complementary method to photoacoustics for estimating energy storage and its time variation, particularly under controlled leaf atmosphere.With modulated light-1 (λ>690 nm) the energy storage approached zero and P700 was maximally oxidized. When background light of shorter wavelength (λ<690 nm-light-2) was added, energy storage momentarily increased (a manifestation of Emerson enhancement) while P700 was reduced. The values of both parameters varied as a function of the background light intensity, keeping a mutual linear relationship. Following the initial change, there was a slow reversal transient of P700 oxidation with a parallel decrease in energy storage. Temporal correlation to P700 redox state after dark adaptation was observed also for the energy storage measured in modulated light 2 when combined with background actinic light of medium intensity (about 50 W m(2)). Under these circumstances P700 was almost totally oxidized initially and then gradually reduced while energy storage was initially low and then increased parallel to P700 reduction.A comparison between the maximum energy storage in modulated light 1, enhanced by background light 2, to the energy storage with short wavelength light (where light tends to be more evenly distributed) indicates a comparable contribution to energy storage from each active photosystem. The above experiments indicate that energy storage contribution from PS I is directly related to the extent of openness of its reaction-centers.While some aspects of the data call for more experimentation, these experiments already establish PTR as a valuable method to monitor photosynthetic energy storage activity in vivo, particularly when used simultaneously with other non-invasive methods.

Evidence for a biological role in photosynthesis for cytochrome b-559--a component of photosystem II reaction center.

Absorbance changes in untreated intact leaf discs, produced upon excitation with high-intensity red light, were shown to be due to the photooxidation of cytochrome b-559. At low intensities (<100 W/m2), photooxidation was almost undetectable. Photooxidation occurred with a half-time of 4.3 sec and an extent of 0.64 mol of cytochrome per 320 mol of chlorophyll. Upon transition to darkness, an additional oxidation occurred that exhibited faster kinetics (t/12 < 100 msec) and 0.32 mol of cytochrome was oxidized per 320 mol of chlorophyll. Photooxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and was specifically induced by red light since far-red light did not cause any absorbance decrease. These results suggest that the redox changes of cytochrome b-559 are driven by photosystem II. Photooxidation was increased by 67% and its initial rate was doubled upon incubation of the leaf in carbonylcyanide p-trifluoromethoxy-phenylhydrazone. Exposure of the leaf to mild water stress or mild heat stress resulted in an increase in the extent of photooxidation and in a 6-fold decrease in the rate constant. Mild heat stress also induced a large increase of the rate constant for the dark reduction of the cytochrome. The dependence of photooxidation on high-intensity red light, its inhibition criteria, the fast transient dark oxidation, and enhancement of both photooxidation and dark transient oxidation by treatments that affect Z, the primary donor to P680, indicate that cytochrome b-559 in vivo is involved in cyclic electron flow around photosystem II. Its primary role in photosynthesis is to divert excess photons from a linear to a cyclic electron flow at high light intensities for protection of the D1 and D2 proteins against photodamage. Dark oxidation of the cytochrome is suggested to reflect a second role, that of deactivation of the powerful oxidant Z+ in the dark.

Photoacoustic studies on the dependence of state transitions on grana stacking.

Photoacoustic detection of oxygen evolution and Emerson enhancement in state 1 and state 2 were compared in a tobacco wild type and mutant (Su/su) deficient in chlorophyll. The mutant shows smaller changes in the distribution of excitation energy between the two photosystems than the wild type. Analysis of Emerson enhancement saturation curves indicates that in the mutant which is deficient in grana partitions and shows less stacking, state 1-state 2 transitions reflect changes in the yield of energy transfer from PS II to PS I (spillover). On the other hand, the wild type containing large grana shows changes in absorption cross-sections of the two photosystems upon state transitions. NaF, a specific phosphatase inhibitor, blocks the transition to state 1, indicating that LHC II phosphorylation has a role in excitation energy regulation in both the mutant as well as the wild type. It is demonstrated that N-ethylmaleimide, a specific sulfhydryl reagent, blocks the transition to state 2, suggesting that a disulfide-sulfhydryl redox couple activates the LHC II kinase in vivo.

Increased Tolerance to Photoinhibitory Light in Paraquat-Resistant Conyza bonariensis Measured by Photoacoustic Spectroscopy and CO(2)-Fixation.

Tolerance to photoinhibition was compared between a paraquat-resistant and a sensitive biotype of Conyza bonariensis (L.). Cronq. Photoinhibitory damage was measured as a decrease in oxygen evolution or energy storage using photoacoustic spectroscopy, or as a decrease of (14)CO(2)-fixation. Prior to exposure to high fluence rates, both biotypes had similar quantum yields of oxygen evolution and energy storage. After exposure to high intensity light, the resistant biotype continued to evolve oxygen and to store energy with a high quantum yield while both energy storage and oxygen evolution were severely reduced in the sensitive biotype. CO(2)-fixation was less rapidly inhibited in the resistant biotype compared to the sensitive one. The data show that the paraquat resistant biotype with its high constitutive levels of the chloroplast localized enzymes of the oxygen detoxification pathway, is also partially protected from photoinhibition. This supports the theory that an enhanced radical scavenging system can give temporary protection against photooxidative damage from a variety of sources.

Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions.

A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance.

Photoinhibition in Chlamydomonas reinhardtii: Effect on state transition, intersystem energy distribution and Photosystem I cyclic electron flow.

The energy distribution, state transitions and photosynthetic electron flow during photoinhibition of Chlamydomonas reinhardtii cells have been studied in vivo using photoacoustics and modulated fluorescence techniques. In cells exposed to 2500 W/m(2) light at 21 °C for 90 min, 90% of the oxygen evolution activity was lost while photochemical energy storage as expressed by the parameter photochemical loss (P.L.) at 710-720 nm was not impaired. The energy storage vs. modulation frequency profile indicated an endothermic step with a rate constant of 2.1 ms. The extent of the P.L. was not affected by DCMU but was greatly reduced by DBMIB. The regulatory mechanism of the state 1 to state 2 transition process was inactivated and the apparent light absorption cross section of photosystem II increased during the first 20 min of photoinhibition followed by a significant decrease relative to that of photosystem I. These results are consistent with the inactivation of the LHC II kinase and the presence of an active cyclic electron flow around photosystem I in photoinhibited cells.

Pulsed photoacoustic detection of flash-induced oxygen evolution from intact leaves and its oscillations.

Photoacoustic signals from intact leaves, produced upon excitation with single-turnover flashes, were shown to be dependent on their position in the flash sequence. Compared to the signal obtained from the first flash, all the others were time-shifted and had increased amplitudes. The signal from the third flash had the largest deviation, whereas that from the second flash deviated only minimally. The amplitude difference of the signals relative to that from the first flash was measured at a convenient time point (5 ms) and showed oscillations of period 4, similar to the O(2)-evolution pattern from algae. These oscillations were strongly damped, tending to a steady state from about the seventh flash on. The extra photoacoustic signal (relative to the first flash) was shown to be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, heat treatment, or water infiltration. Its change with flash number, its saturation with increasing flash energy, and the above inhibition criteria indicate that it originates in pulsed O(2) evolution. The sound wave produced by the first flash, however, arose by a photothermal mechanism only, as shown by its linear dependence on the flash intensity and insensitivity to the above treatments. The above flash pattern demonstrates that the photocycle of the S states (i.e., positive charge accumulation before two water molecules can be oxidized in a concerted way to produce molecular oxygen) occurs in intact leaves. It proves the applicability of the photoacoustic method for mechanistic studies of O(2) evolution in leaves under physiological conditions. Water content of leaves is readily measured by this method.

Role of the cytochrome b6.f complex in the redox-controlled activity of Acetabularia thylakoid protein kinase.

The regulation of the protein kinase activity responsible for the phosphorylation of the light-harvesting complex of photosystem II (LHCII) 27-kDa polypeptide involved in the State I-State II transitions in Acetabularia thylakoids was investigated. The LHCII kinase of isolated thylakoids retains its activity in absence of light-driven electron flow or reductants added in the dark. However, the kinase is reversibly inactivated by addition of oxidants in vitro or by far red (710 nm) light in vivo. Inhibitors of the quinol oxidase site of the cytochrome b6.f complex inactivate the LHCII kinase in the dark, and also in the light, or in presence of duroquinol when the plastoquinone pool is reduced. Inhibitors of the quinone reductase site of the b6.f complex have practically no effect in the dark and stimulate the kinase activity in the light. Based on these data and on our previous report, showing specific loss of LHCII kinase activity in a Lemna mutant lacking the cytochrome b6.f complex (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210), we propose that the activity of the LHCII kinase is regulated by the redox state of a cytochrome b6.f complex component(s) which responds to the balance of electron flow from photosystem II via the plastoquinone pool to photosystem I.

Photosynthetic responses of leaves to water stress, expressed by photoacoustics and related methods : I. Probing the photoacoustic method as an indicator for water stress in vivo.

The effect of leaf desiccation on the photosynthetic activities in vivo was probed by the photoacoustic method. The aim of this research was: (a) To study the photoacoustic signal per se in varied conditions in order to develop this tool as a probe for stress conditions in vivo. (b) To obtain results pertaining to electron transport activities in vivo, and confirm conclusions based on work with isolated chloroplasts, which could otherwise be the result of nonspecific damage occurring during their isolation. Leaf discs from tobacco (Nicotiana tabacum L.) were routinely used, with other species tested also for comparison. Rapid leaf desiccation caused changes in the low frequency photoacoustic signal, attributed both to the mechanism of signal transduction, influenced by changes in the structural parameters of the leaf, and to the direct (nonstomatal) inhibition of gross photosynthesis. The dependence of the photothermal part of the signal on the frequency indicated the presence of two photothermal components, one of which persisted only at low modulation frequencies (below about 100 Hz) and which largely increased with the desiccation treatment. This component was ascribed to a thermal wave which reaches the leaf surface. The other nonvariable photothermal component was ascribed to a thermal wave propagating from the chloroplasts to the surface of the mesophyll cell. Only this component is considered in the ratio of the O(2) signal to the photothermal signal, which is used to estimate the quantum yield of photosynthesis. The specific dependence of the latter ratio on the frequency yielded a comparative quantum yield parameter from its extrapolation to zero frequency, and also indicated stress induced changes in the diffusion of O(2) through the mesophyll cell, reflected by changes in its characteristic slope. The (zero frequency extrapolated) quantum yield was markedly reduced with the progression of the water stress, indicating the inhibition of (gross) phototosynthetic electron transport in vivo. This result was expressed even more emphatically by the stronger inhibition of the photochemical energy storage, obtained by photoacoustic measurements at a high modulation frequency.

Photosynthetic Responses of Leaves to Water Stress, Expressed by Photoacoustics and Related Methods : II. The Effect of Rapid Drought on the Electron Transport and the Relative Activities of the Two Photosystems.

The effect of rapid dehydration of detached tobacco leaves (Nicotiana tabacum L.) on the photochemical apparatus of photosynthesis was studied in vivo by a combination of methods: photoacoustics, chlorophyll a fluorescence, and cytochrome f difference spectroscopy. It was shown that the inhibition of gross O(2) evolution was mainly caused by inactivation of PSII: (a) The saturation curve of cytochrome-f photooxidation by farred (>710 nanometers) light was resistant to the stress, leading to the conclusion that photosystem I (PSI) was largely unaffected by the stress. (b) The extent of the chlorophyll a variable fluorescence arising from photosystem II (PSII) decreased with the progression of the stress, but was largely unaffected when the leaf was preincubated with electron donors to PSII, such as hydroxylamine. It is concluded that the drought damage to PSII occurred on the photooxidative side. Despite the extensive inhibition of PSII and the relative preservation of PSI, the apparent PSII/PSI activity balance was somewhat larger in stressed leaves than in the control, as indicated by photoacoustic measurements of Emerson enhancement. These measurements were performed continuously under conditions which favor transitions to either state 1 or 2, showing that the transition to state 2 was considerably inhibited. Simultaneous measurements of chlorophyll fluorescence induction at 680 and 730 mm at room temperature were also used to probe changes in energy distribution between PSII and PSI and indicated that the transition from a dark adapted state to state 2 was also affected in water-stressed leaves. The saturation curve of the far-red light effect in Emerson enhancement was not changed by the stress, giving another independent evidence for the drought resistance of PSI activity. This apparent preservation of the imbalance in photochemical activities in favor of PSII, despite the fact that PSII is strongly inhibited, and PSI is not, supports a previous suggestion that the electron transfer between the two photosystems is not random but that a large extent of PSII and PSI units are specifically linked.

Analysis of Emerson enhancement under conditions where photosystem II is inhibited - Are the two photosystems indeed separated?

Photoacoustic measurements of photosynthetic oxygen evolution and its enhancement by addition of background far-red light (Emerson enhancement) were made on both intact and inhibited or stressed leaves. The extent of enhancement increased with the dehydration treatment and decreased with a mild heat treatment or with the addition of DCMU. It nevertheless persisted even at a very high degree of inhibition - a result which indicates high population ratio of single pairs of combined photosystem II and photosystem I units, functional in whole chain electron transport. This implies a restriction on the separation between photosystems I and II, in contrast to existing concepts.

Comparison of photosynthetic parameters of an aurea mutant (Su/su) of tobacco and the wild-type by the photoacoustic method.

Oxygen evolution and energy storage yields in tobacco (Nicotiana tabacum L.) wild-type (cv. John Williams Broadleaf) and a mutant (Su/su) deficient in chlorophyll were compared using the photoacoustic technique. Oxygen-evolution and energy-storage quantum yields in the mutant were higher when measured in red light (640-690 nm) than green or blue light (540 nm and 440 nm, respectively), indicating that carotenoids in this mutant do not transfer energy efficiently to the photochemical reaction centers. It is suggested that carotenoids may play a role in protecting the photosynthetic apparatus against damage by high energy fluxes. In the wild-type, the oxygenevolution yield did not change drastically throughout the visible spectrum. The mutant had a higher quantum yield of oxygen evolution than the wildtype. Similarly maximum rates obtained from saturation curves for the mutant were more than twice higher per leaf area and about five times higher per chlorophyll, as compared to the wild-type.