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Certain introns significantly increase mRNA accumulation by a poorly understood mechanism. These introns have no effect when located upstream, or more than ~1 Kb downstream, of the start of transcription. We tested the ability of a formerly non-stimulating intron containing 11 copies of the sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, to affect expression from various positions. The activity profile of this intron at different locations was similar to that of a natural intron from the UBQ10 gene, suggesting that the motif increases mRNA accumulation by the same mechanism. A series of introns with different numbers of this motif revealed that the effect on expression is linearly dependent on motif copy number up to at least 20, with each copy adding another 1.5-fold increase in mRNA accumulation. Furthermore, 6 copies of the motif stimulated mRNA accumulation to a similar degree from within an intron or when introduced into the 5′-UTR and coding sequences of an intronless construct, demonstrating that splicing is not required for this sequence to boost expression. The ability of this motif to substantially elevate expression from several hundred nucleotides downstream of the transcription start site reveals a novel type of eukaryotic gene regulation.

A picture is beginning to emerge from a variety of organisms that for a subset of genes, the most important sequences that regulate expression are situated not in the promoter but rather are located within introns in the first kilobase of transcribed sequences. The actual sequences involved are difficult to identify either by sequence comparisons or by deletion analysis because they are dispersed, additive, and poorly conserved. However, expression-controlling introns can be identified computationally in species with relatively small introns, based on genome-wide differences in oligomer composition between promoter-proximal and distal introns. The genes regulated by introns are often expressed in most tissues and are among the most highly expressed in the genome. The ability of some introns to strongly stimulate mRNA accumulation from several hundred nucleotides downstream of the transcription start site, even when the promoter has been deleted, reveals that our understanding of gene expression remains incomplete. It is unlikely that any diseases are caused by point mutations or small deletions that reduce the expression of an intron-regulated gene unless splicing is also affected. However, introns may be particularly useful in practical applications such as gene therapy because they strongly activate expression but only affect the transcription unit in which they are located.

Certain introns strongly increase mRNA accumulation by a poorly understood mechanism known as Intron-Mediated Enhancement (IME). Introns that boost expression by IME have no effect when located upstream of or more than ~1 Kb downstream from the start of transcription. The sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, can convert a non-stimulating intron into one that strongly increases mRNA accumulation. We tested the ability of an intron containing this motif to stimulate expression from different locations and found that it had the same positional requirements as naturally occurring IME introns. The motif also stimulated gene expression from within the 5’-UTR and coding sequences of an intronless construct. Furthermore, the 5’-UTR of another gene increased expression when inserted into an otherwise non-stimulating intron in coding sequences. These results demonstrate that splicing is not required for intron-mediated enhancement, and suggest that other sequences downstream of the transcription start site in addition to introns may stimulate expression by a similar mechanism.

Within two years of their discovery in 1977, introns were found to have a positive effect on gene expression. Numerous examples of stimulatory introns have been described since then in very diverse organisms, including plants. In some cases, the mechanism through which the intron affects expression is readily understood. However, many introns that affect expression increase mRNA accumulation through an unknown mechanism, referred to as intron-mediated enhancement (IME). Despite several decades of research into IME, and the clear benefits of using introns to increase transgene expression, little progress has been made in understanding the mechanism of IME. Several fundamental questions regarding the role of transcription and splicing, the sequences responsible for IME, the involvement of other factors, and the relationship between introns and promoters remain unanswered. The more we learn about the properties of stimulating introns, the clearer it becomes that the effects of introns are unfamiliar and difficult to reconcile with conventional views of how transcription is controlled. We hypothesize that introns increase transcript initiation upstream of themselves by creating a localized region of accessible chromatin. Introns might represent a novel kind of downstream regulatory element for genes transcribed by RNA polymerase II.

To more precisely define the positions from which certain intronic regulatory sequences increase mRNA accumulation, the effect of a UBIQUITIN intron on gene expression was tested from six different positions surrounding the transcription start site (TSS) of a reporter gene fusion in Arabidopsis thaliana The intron increased expression from all transcribed positions but had no effect when upstream of the 5'-most TSS. While this implies that the intron must be transcribed to increase expression, the TSS changed when the intron was located in the 5'-untranslated region (UTR), suggesting that the intron affects transcription initiation. Remarkably, deleting 303 nucleotides of the promoter including all known TSSs and all but 18 nucleotides of the 5'-UTR had virtually no effect on the level of gene expression as long as an intron containing stimulatory sequences was included. Instead, transcription was initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. These results suggest that certain intronic DNA sequences play unexpectedly large roles in directing transcription initiation and constitute a previously unrecognized type of downstream regulatory element for genes transcribed by RNA polymerase II.

Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated. To test the effects of the same intron on the expression of a range of genes, GUS translational fusions were constructed using the promoters of eight Arabidopsis genes whose expression was reported to be constitutive (GAE1, CNGC2 and ROP10), tissue specific (ADL1A, YAB3 and AtAMT2) or regulated by light (ULI3 and MSBP1). For each gene, a fusion containing the first intron from the UBQ10 gene was compared to fusions containing the gene's endogenous first intron (if the gene has one) or no intron. In every case, the UBQ10 intron increased expression relative to the intronless control, although the magnitude of the change and the level of expression varied. The UBQ10 intron also changed the expression patterns of the CNGC2 and YAB3 fusions to include strong activity in roots, indicating that tissue specificity was disrupted by this intron. In contrast, the regulation of the ULI3 and MSBP1 genes by light was preserved when their expression was stimulated by the intron. These findings have important implications for biotechnology applications in which a high level of transgene expression in only certain tissues is desired.

In diverse eukaryotes, certain introns increase mRNA accumulation through the poorly understood mechanism of intron-mediated enhancement (IME). A distinguishing feature of IME is that these introns have no effect from upstream or more than 1 Kb downstream of the transcription start site (TSS). To more precisely define the intron position requirements for IME in Arabidopsis, we tested the effect of theUBQ10intron on gene expression from 6 different positions surrounding the TSS of aTRP1:GUSfusion. The intron strongly increased expression from all transcribed positions, but had no effect when 204 nt or more upstream of the 5’-most TSS. When the intron was located in the 5’ UTR, the TSS unexpectedly changed, resulting in longer transcripts. Remarkably, deleting 303 nt of the core promoter, including all known TSS’s and all but 18 nt of the 5’ UTR, had virtually no effect on the level of gene expression as long as a stimulating intron was included in the gene. When the core promoter was deleted, transcription initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. Together, these results suggest that certain introns play unexpectedly large roles in directing transcription initiation and represent a previously unrecognized type of downstream regulatory elements for genes transcribed by RNA polymerase II. This study also demonstrates considerable flexibility in the sequences surrounding the TSS, indicating that the TSS is not determined by promoter sequences alone. These findings are relevant in practical applications where introns are used to increase gene expression and contribute to our general understanding of gene structure and regulation in eukaryotes.

Related motifs strongly increase gene expression when added to an intron located in coding sequences. Many introns greatly increase gene expression through a mechanism that remains elusive. An obstacle to understanding intron-mediated enhancement (IME) has been the difficulty of locating the specific intron sequences responsible for boosting expression because they are redundant, dispersed, and degenerate. Previously we used the IMEter algorithm in two independent ways to identify two motifs (CGATT and TTNGATYTG) that are candidates for involvement in IME in Arabidopsis. Here we show that both motifs are sufficient to increase expression. An intron that has little influence on expression was converted into one that increased mRNA accumulation 24-fold and reporter enzyme activity 40-fold relative to the intronless control by introducing 11 copies of the more active TTNGATYTG motif. This degree of stimulation is twice as large as that of the strongest of 15 natural introns previously tested in the same reporter gene. Even though the CGATT and TTNGATYTG motifs each increased expression, and CGATT matches the NGATY core of the longer motif, combining the motifs to make TTCGATTTG reduced the stimulating ability of the TTNGATYTG motif. Additional substitutions were used to test the contribution to IME of other residues in the TTNGATYTG motif. The verification that these motifs are active in IME will improve our ability to predict the stimulating ability of introns, to engineer any intron to increase expression to a desired level, and to explore the mechanism of IME by seeking factors that might interact with these sequences.

Introns that elevate mRNA accumulation have been found in a wide range of eukaryotes. However, not all introns affect gene expression, and direct testing is currently the only way to identify stimulatory introns. Our genome-wide analysis in Arabidopsis thaliana revealed that promoter-proximal introns as a group are compositionally distinct from distal introns and that the degree to which an individual intron matches the promoter-proximal intron profile is a strong predictor of its ability to increase expression. We found that the sequences responsible for elevating expression are dispersed throughout an enhancing intron, as is a candidate motif that is overrepresented in first introns and whose occurrence in tested introns is proportional to its effect on expression. The signals responsible for intron-mediated enhancement are apparently conserved between Arabidopsis and rice (Oryza sativa) despite the large evolutionary distance separating these plants.

The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.

Introns are often required for full expression of genes in organisms as diverse as plants, insects, nematodes, yeast, and mammals. To explore the potential mechanisms of intron-mediated enhancement in Arabidopsis thaliana, the effect of varying the position of an intron was determined using a series of reporter gene fusions between TRYPTOPHAN BIOSYNTHESIS1 (TRP1) and GUS. Two introns that differ in the degree to which they stimulate expression were individually tested at six locations within coding sequences and two positions in the 3'-UTR. The ability of the first introns from both the TRP1 and POLYUBIQUITIN10 (UBQ10) genes to elevate mRNA accumulation in transgenic plants was found to decline with distance from the promoter, despite their being efficiently spliced from all coding sequence locations. Neither intron significantly enhanced mRNA accumulation when positioned 1.1 kb or more from the start of transcription. In addition, measurements of GUS enzyme activity revealed that both introns at all locations elevated GUS activity more than they enhanced mRNA accumulation. The stimulation mediated by two of four other introns tested at the position nearest the promoter was also greater at the level of GUS activity than mRNA accumulation. These findings support a model in which introns increase transcription and promote translation by two distinct mechanisms.

The composition of humid, unfertilised, short‐tussock (Festuca‐Rytidosperma‐Poa) grasslands was recorded on 42 permanent transects in 1959, 1970–72, and 1995–96 in the Wairau catchment, Marlborough. Changes in frequency of the most abundant plant species and bare ground were analysed. Reduced levels of grazing, browsing, and fire prompted significant recovery of native shrubs, tall tussocks (Chionochloa), and herbs. Invasive exotics (Hieracium, Anthoxanthum, Agrostis) also increased. Short native grasses (Poa, Elymus), a native herb (Acaena spp.), and an exotic herb (Rumex acetosella) declined. Hieracium pilosella, H. caespitosum, and H. lepidulum were rare or absent in 1959. By 1996 they had established on half the transects, but were still in the early stages of invasion (median frequencies ≤ 5%). There was no correlation between the increase in Hieracium and decline in other species, suggesting that other factors were driving the compositional changes. However, Hieracium species can establish ...

To investigate how plants acquire and assimilate sulfur from their environment, we isolated and characterized two mutants of Arabidopsis thaliana deficient in sulfate transport. The mutants are resistant to selenate, a toxic analogue of sulfate. They are allelic to each other and to the previously isolated sel1 (selenate-resistant) mutants, and have been designated sel1-8 and sel1-9. Root elongation in these mutants is less sensitive to selenate than in wild-type plants. Sulfate uptake into the roots is impaired in the mutants under both sulfur-sufficient and sulfur-deficient conditions, but transport of sulfate to the shoot is not affected. The sel1 mutants contain lesions in the sulfate transporter gene Sultr1;2 located on the lower arm of chromosome 1. The sel1-1, sel1-3 and sel1-8 mutants contain point mutations in the coding sequences of Sultr1;2, while the sel1-9 mutant has a T-DNA insertion in the Sultr1;2 promoter. The Sultr1;2 cDNA derived from wild-type plants is able to complement Saccharomyces cerevisiae mutants defective in sulfate transport, but the Sultr1;2 cDNA from sel1-8 is not. The Sultr1;2 gene is expressed mainly in roots, and accumulation of transcripts increases during sulfate deprivation. Examination of transgenic plants containing the Sultr1;2 promoter fused to the GUS-reporter gene indicates that Sultr1;2 is expressed mainly in the root cortex, the root tip and lateral roots. Weaker expression of the reporter gene was observed in hydathodes, guard cells and auxiliary buds of leaves, and in anthers and the basal parts of flowers. The results indicate that Sultr1;2 is primarily involved in importing sulfate from the environment into the root.

The distribution and density of Hieracium pilosella, H. caespitosum, and H. lepi‐dulum seedlings were quantified at an early stage of invasion of a tall‐tussock (Chionochloa) and a more open short‐tussock (Festuca‐Poa) grassland. Seedling and available (unoccupied) microsites were compared in 11 stands. Recorded variables included establishment substrate, overhanging cover, and proximity and height of neighbouring plants. Total Hieracium cover was

The properties of Penicillium chrysogenum adenosine 5′-phosphosulfate (APS) kinase mutated at Ser-107 were examined. Ser-107 is analogous to a serine of the E. coli enzyme that has been shown to serve as an intermediate acceptor in the transfer of a phosphoryl group from ATP to APS. Replacement of Ser-107 with alanine yielded an active enzyme with kinetic characteristics similar to those of wild-type APS kinase. Another mutant form of the enzyme in which Ser-107 was replaced by cysteine was also active. Covalent modification of Cys-107 eliminated catalytic activity, and substrates protected against modification. Mutation of Ser-97, of Ser-99, of Thr-103, of Ser-104 to alanine, or of Tyr-109 to phenylalanine also yielded an active enzyme. The cumulative results indicate that Ser-107 may reside in the substrate binding pocket of fungal APS kinase, but neither it nor any nearby hydroxy amino acid serves as an obligatory phophoryl acceptor in the 3′-phosphoadenylylsulfate synthesis reaction. The results also indicate that the absence of a serine at position 478 in the APS kinase-like C-terminal region of fungal ATP sulfurylase does not account for the lack of APS kinase activity in that enzyme. However, mutating the ATP P-loop residues in APS kinase to those found in the analogous C-terminal region of fungal ATP sulfurylase eliminated enzyme activity.

Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic selections and fluorescence screens. Each was shown to contain a recessive allele of trp1, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trp1 mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trp1 alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trp1 mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.

Many introns significantly increase gene expression through a process termed intron-mediated enhancement (IME). Introns exist in the transcribed DNA and the nascent RNA, and could affect expression from either location. To determine which is more relevant to IME, hybrid introns were constructed that contain sequences from stimulating Arabidopsis thaliana introns either in their normal orientation or as the reverse complement. Both ends of each intron are from the non-stimulatory COR15a intron in their normal orientation to allow splicing. The inversions create major alterations to the sequence of the transcribed RNA with relatively minor changes to the DNA structure. Introns containing portions of either the UBQ10 or ATPK1 intron increased expression to a similar degree regardless of orientation. Also, computational predictions of IME improve when both intron strands are considered. These findings are more consistent with models of IME that act at the level of DNA rather than RNA.

Introns in a wide range of organisms including plants, animals and fungi are able to increase the expression of the gene that they are contained in. This process of intron-mediated enhancement (IME) is most thoroughly studied in Arabidopsis thaliana, where it has been shown that enhancing introns are typically located near the promoter and are compositionally distinct from downstream introns. In this study, we perform a comprehensive comparative analysis of several sequenced plant genomes. We find that enhancing sequences are conserved in the multi-cellular plants but are either absent or unrecognizable in algae. IME signals are preferentially located towards the 5′-end of first introns but also appear to be enriched in 5′-UTRs and coding regions near the transcription start site. Enhancing introns are found most prominently in genes that are highly expressed in a wide range of tissues. Through site-directed mutagenesis in A. thaliana, we show that IME signals can be inserted or removed from introns to increase or decrease gene expression. Although we do not yet know the specific mechanism of IME, the predicted signals appear to be both functional and highly conserved.

Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression. The IMEter demonstrates that introns located near the beginning of genes are compositionally distinct from later introns and that these differences are closely related to the ability of some introns to increase gene expression. This word-based approach has proven more successful than deletion analysis at identifying the sequences responsible for elevating expression because they are dispersed throughout stimulatory introns.

Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression. The IMEter demonstrates that introns located near the beginning of genes are compositionally distinct from later introns and that these differences are closely related to the ability of some introns to increase gene expression. This word-based approach has proven more successful than deletion analysis at identifying the sequences responsible for elevating expression because they are dispersed throughout stimulatory introns.

Tree size and age structures, treefall and canopy gap characteristics, and regeneration responses to treefalls were compared for three stands of old-growth beech (Notho- fagus) forest dominated by N. fusca and N. menziesii on the South Island, New Zealand. Treefall gaps (up to 1000 m2) were most often caused by standing trees killed by drought and/or insect attack, or by trees snapped by wind. The causes of gap formation and the size and age distributions of treefall gaps varied between localities because of spatial and temporal differences in the histories of disturbance. At Fergies Bush where drought-related dieback had produced many large gaps with standing dead trees, gaps were generally young. Con- versely, at Station Creek, small, old gaps formed by bolesnap dominated the disturbance regime. At Rough Creek, gaps of all ages and sizes were found along with an almost complete fern cover, and abundant shrubs and occasional subcanopy hardwood trees. Although overall patterns of regeneration were unrelated to differences in gap size, the relative abundance of N. fusca and N. menziesii varied between localities according to the seemingly minor differences in forest structure and disturb- ance history described above. Interpretations of regeneration response to gap parameters, therefore, need to account for differences in disturbance history between sites. Differences in the disturbance history between localities will also influ- ence rates of gap closure, and because closure rates are used to estimate forest turnover times, meaningful comparisons of disturbance regimes for different forest types can only be made if this intersite variability is addressed.

We describe a reproducible protocol for the analysis of individual members of complex mammalian gene families by gel fractionation in two dimensions within a specially designed, easily built electrophoretic apparatus. We have used this protocol to resolve the family of mouse H-2 class I genes, with approximately 30 members, as well as two different families of endogenous retroviral-like sequences, each of which has approximately 180 members dispersed throughout the genome. The results demonstrate the feasibility of using this protocol for rapid, whole genome analysis of individual animals and cell lines. Two-dimensional DNA analysis of highly repeated retroviral-like DNA families could be applied to genetic mapping and cloning experiments as well as to obtaining whole genome fingerprints in the analysis of somatic cell hybrid lines that contain a subset of chromosomes from the genome of interest.

Introns can significantly affect gene expression in plants and many other eukaryotes in a variety of ways. Several types of gene regulation, both positive and negative, that involve plant introns are reviewed in this chapter. Some introns contain enhancer elements or alternative promoters, while many others elevate mRNA accumulation by a different process that has been named intron-mediated enhancement (IME). The introns involved in IME must be within transcribed sequences near the start of a gene and in their natural orientation to increase expression. The intron sequences involved are still poorly defined, and the mechanism of IME remains mysterious. A model of IME is presented in which introns increase transcript elongation.

Size and age structure analysis, dated past disturbances, treefall replacement patterns, and spatial pattern analysis were used to reconstruct the developmental history of two old-growth Nothofagus fusca/N. menziesii stands, South Island, New Zealand. Diameter and height class distributions suggested that N. menziesii was replacing N. fusca, however, stand history reconstruction analysis showed that both species had regenerated intermittently after small-scale disturbances. Although large-scale disturbances such as blowdowns may occasionally generate even-aged stands, gap-phase regeneration maintains the forests in compositional equilibrium. In the absence of other competing tree species and understorey plants the two species appear to coexist by way of different life history strategies, where one species (N. menziesii) has low juvenile mortality and the other (N. fusca) has faster height growth rates and greater longevity and adult survivorship.

Whereas many important biological discoveries have been made using plants, subsequent progress in some areas of plant research has fallen behind that in other organisms for which funding and in vitro assays are more readily available. Gene expression is one such field in which importance continues to grow because many potential plant biotechnology–based solutions to global problems depend on regulating the expression of specific genes. Previous limitations to exploring gene expression in plants have been partially mitigated by recent advances in genomics, genetics, and transformation techniques. The book Regulation of Gene Expression in Plants: The Role of Transcript Structure and Processing , edited by Carole L. Bassett, summarizes our current understanding of plant gene expression, with an emphasis on transcriptional and posttranscriptional regulation. The topics covered in six chapters include differences in messenger RNA (mRNA) structure caused by variations in transcription start and polyadenylation sites, alternative splicing, regulation by small RNAs, and mRNA transport and degradation. The chapters vary in depth, quality, and the degree to which the emphasis is placed on plants rather than eukaryotes in general. However, this slim volume is a useful review of gene expression in plants. The question of whether or not all differences in mRNA structure have functional importance remains open.

Introns are often required for full expression of genes in organisms as diverse as plants, insects, nematodes, yeast, and mammals. To explore the potential mechanisms of intron-mediated enhancement in Arabidopsis thaliana, the effect of varying the position of an intron was determined using a series of reporter gene fusions between TRYPTOPHAN BIOSYNTHESIS1 (TRP1) and GUS. Two introns that differ in the degree to which they stimulate expression were individually tested at six locations within coding sequences and two positions in the 3'-UTR. The ability of the first introns from both the TRP1 and POLYUBIQUITIN10 (UBQ10) genes to elevate mRNA accumulation in transgenic plants was found to decline with distance from the promoter, despite their being efficiently spliced from all coding sequence locations. Neither intron significantly enhanced mRNA accumulation when positioned 1.1 kb or more from the start of transcription. In addition, measurements of GUS enzyme activity revealed that both introns at all locations elevated GUS activity more than they enhanced mRNA accumulation. The stimulation mediated by two of four other introns tested at the position nearest the promoter was also greater at the level of GUS activity than mRNA accumulation. These findings support a model in which introns increase transcription and promote translation by two distinct mechanisms.

To explore possible mechanisms of intron-mediated enhancement of gene expression, the features of PAT1 intron 1 required to elevate mRNA accumulation were systematically tested in transgenic Arabidopsis. This intron is remarkably resilient, retaining some ability to increase mRNA accumulation when splicing was prevented by mutation of 5' and 3' splice sites, branchpoint sequences, or when intron U-richness was reduced. Enhancement was abolished by simultaneously eliminating branchpoints and the 5' splice site, structures involved in the first two steps of spliceosome assembly. Although this suggests that the splicing machinery is required, intron splicing is clearly not enough to enhance mRNA accumulation. Five other introns were all efficiently spliced but varied widely in their ability to increase mRNA levels. Furthermore, PAT1 intron 1 was spliced but lost the ability to elevate mRNA accumulation when moved to the 3' UTR. These findings demonstrate that splicing per se is neither necessary nor sufficient for an intron to enhance mRNA accumulation, and suggest a mechanism that requires intron recognition by the splicing machinery but also involves nonconserved intron sequences.

Summary The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

The SIR gene products maintain transcriptional repression at the silent mating type loci and telomeres in Saccharomyces cerevisiae, although no enzymatic or structural activity has been assigned to any of the Sir proteins nor has the role of any of these proteins in transcriptional silencing been clearly defined. We have investigated the functions and interactions of the Sir2, Sir3, and Sir4 proteins by overexpressing them in yeast cells. We find that Sir2p and Sir3p are toxic when overexpressed, while high Sir4p levels have no toxic effect. Epistasis experiments indicate that Sir2p-induced toxicity is diminished in strains lacking the SIR3 gene, while both Sir2p and Sir4p are required for Sir3p to manifest its full toxic effect. In addition, the effects of Sir2 or Sir3 overexpression are exacerbated by specific mutations in the N-terminus of the histone H4 gene. These results are consistent with a model in which Sir2p, Sir3p and Sir4p function as a complex and interact with histones to modify chromatin structure. We find no evidence that toxicity from high levels of the Sir proteins results from widespread repression of transcription. Instead, we find that high levels of Sir2p and/or Sir3p cause a profound decrease in chromosome stability. These results can be appreciated in the context of the effects of Sir2p in histone acetylation and of chromatin structure on chromosome stability.

A long term goal for this laboratory is to obtain a detailed understanding of the biochemical and genetic mechanisms that plants use to regulate the synthesis of the amino acid tryptophan (Last et al., 1992; Last, 1993). In addition to its role in the production of tryptophan for protein synthesis, this pathway gives rise to many indolic secondary metabolites, including the auxin, indole-3-acetic acid (Wright et al., 1991) and the Arabidopsis phytoallexin called camalexin (Tsuji et al., 1992). Thus, we anticipate that plants regulate the enzymes of tryptophan biosynthesis in response to requirements for both protein and secondary product biosynthesis.

Two classes of sequences in the yeast Saccharomyces cerevisiae are subject to transcriptional silencing: the silent mating-type cassettes and telomeres. In this report we demonstrate that the silencing of these regions is strictly associated with acetylation of the epsilon-amino groups of lysines in the amino-terminal domains of three of the four core histones. Both the silent mating-type cassettes and the Y domains of telomeres are packaged in nucleosomes in vivo that are hypoacetylated relative to those packaging active genes. This difference in acetylation is eliminated by genetic inactivation of silencing: The silent cassettes from sir2, sir3, or sir4 cells show the same level of acetylation as other active genes. The correspondence of silencing and hypoacetylation of the mating-type cassettes is observed even for an allele lacking a promoter, indicating that silencing per se, rather than the absence of transcription, is correlated with hypoacetylation. Finally, overexpression of Sir2p, a protein required for transcriptional silencing in yeast, yields substantial histone deacetylation in vivo. These studies fortify the hypothesis that silencing in yeast results from heterochromatin formation and argue that the silencing proteins participate in this formation.

An Arabidopsis thaliana gene encoding phosphoribosylanthranilate transferase is shown to be the gene that is defective in blue fluorescent trp1 mutant plants. This gene, named PAT1, was isolated using an A. thaliana cDNA clone that suppressed an Escherichia coli trpD(-) mutation. The PAT1 coding region is homologous to those for the phosphoribosylanthranilate transferases from many microorganisms. Unlike other genes involved in aromatic amino acid biosynthesis in A. thaliana, PAT1 appears to be a single-copy gene. PAT1 was demonstrated to be the gene that is defective in blue fluorescent trp1 mutants by two methods: genetic complementation in transgenic plants and genetic mapping studies. This is the first report of cloning a plant phosphoribosylanthranilate transferase gene. The PAT1 gene should prove useful as a selectable marker for transformation or a visible reporter of gene expression when used in conjunction with trp1 plants.

Age-states, population structure, and seedling regeneration of the grass Chionochloa pallens were in- vestigated in eight alpine grassland stands representing the range of canopy cover (ca. 5 - 75 %) and basal area (112 - 3889 m2/ha-1) in the headwaters of the Avoca catchment, Canterbury, New Zealand. In open-canopy stands (< 50 % canopy cover, < 2000 m2/ha-1 basal area) C. pallens individuals were clearly distinguishable. Rela- tionships between basal diameter, numberof tillers, height, and % crown death suggested four putative age-states: 'seedlings', 'juvenile tussocks', 'mature tussocks' and 'senescent tussocks'. Four types of population structure were identified depending on the proportions of seedlings and juvenile and mature tussocks. C. pallens seedlings were present in seven stands at densities ranging from 0.6 to 123.4 /m2. They were most frequent on microsites pro- tected from frost-heave by short herbaceous vegetation or litter, but with little competition from taller vegetation. Differences in seedling densities between stands partly reflected the proximity and abundance of seed sources and degree of protection from frost-heave. C. pallens population structures appear to be influenced by the fre- quency and magnitude of geomorphic disturbances and browsing by introduced animals.

The product of the Saccharomyces cerevisiae SIR4 gene, in conjunction with at least three other gene products, prevents expression of mating-type genes resident at loci at either end of chromosome III, but not of the same genes resident at the MAT locus in the middle of the chromosome. To address the mechanism of this novel position effect regulation, we have conducted a structural and genetic analysis of the SIR4 gene. We have determined the nucleotide sequence of the gene and found that it encodes a lysine-rich, serine-rich protein of 152 kilodaltons. Expression of the carboxy half of the protein complements a chromosomal nonsense mutation of sir4 but not a complete deletion of the gene. These results suggest that SIR4 protein activity resides in two portions of the molecule, but that these domains need not be covalently linked to execute their biological function. We also found that high-level expression of the carboxy domain of the protein yields dominant derepression of the silent loci. This anti-Sir activity can be reversed by increased expression of the SIR3 gene, whose product is normally also required for maintaining repression of the silent loci. These results are consistent with the hypothesis that SIR3 and SIR4 proteins physically associate to form a multicomponent complex required for repression of the silent mating-type loci.

The his4-917 mutation of yeast results from the insertion of a Ty element, Ty917, into the 5' regulatory region of the HIS4 gene. Ty917 prevents HIS4 transcription, thus rendering the cell histidine requiring. Recombination between Ty917 and a Ty element elsewhere in the yeast genome can result in the replacement of part or all of the Ty917 element by sequences from the Ty element. Recombinant derivatives display a variety of phenotypes including His-, weakly His+, and strongly His+. In most of the His+ derivatives, the expression of HIS4 is controlled by genes at the mating type locus. To identify the Ty sequences important in controlling the expression of an adjacent gene, we used Ty elements that have different effects on gene expression to construct hybrid Ty elements in vitro. The effects of these hybrid elements on HIS4 expression were examined. These experiments indicate that the critical sequence differences between Ty elements that permit HIS4 expression and those that prevent its expression lie in the rightmost (HIS4-proximal) 730 base pairs of the element. The DNA sequence of this region was determined for three elements: Ty917, which prevents HIS4 expression; Ty917(467), which confers a weak His+ phenotype; and Ty917(480), which confers a strong His+ phenotype. Within this region, Ty917(467) differs from Ty917 by a single base-pair change that is in the internal (epsilon) region of the Ty element. Ty917(480) differs from Ty917 by this same base-pair change and by 10 changes in the terminal delta sequence. The sequence change common to Ty917(467) and Ty917(480) lies in a region of the Ty element that is homologous to the simian virus 40 enhancer of transcription.

Size and age structure analysis were used to reconstruct the population dynamics of six apparently old and stable stands dominated by the conifers Libocedrus bidwillii and/or Dacrydium biforme in Camp Creek, West land, New Zealand. All stands contained relatively even-aged groups of Libocedrus that had probably originat ed after either mass movement or windthrow. The age distributions of stands reflected the effects of distur bances 200-400 yr ago. There was no evidence to support synchronous failure of regeneration of Libocedrus and Dacrydium bi forme. Between 1600 and 1800 A.D., the period of the postulated conifer regeneration gap, over 70% of trees of these species had established. A lack of Libocedrus regeneration over the last 200 yr reflects a lack of distur bance over that time. Because of the dominating influence of disturbances and other factors it may be inap propriate to use tree population structures alone to substantiate or refute claims of recent climate change.

The Ty (transposon yeast) elements of Saccharomyces cerevisiae comprise a family of dispersed, repeated DNA sequences (see Ref. 11, 13, and 19 for reviews). Each element consists of an internal region, referred to as epsilon, flanked by direct terminal repeats known as delta sequences. The epsilon region is approximately 5.4 kbp in length, and the delta sequences are about 335 bp long. The Ty elements display considerable sequence divergence. In general, the elements can be divided into two classes known as Ty1 and Ty2; these classes differ from each other by two large blocks of heterologous sequence. In the haploid genomes of most laboratory strains of yeast, there are 25 to 30 Ty1 elements and five to six Ty2 elements.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, mediated independently through two cis-acting sites, termed the E and I silencers. We have found that in the absence of the I silencer, deletion of any one of three distinct elements within E yields partial derepression of the mating-type genes resident at HML, whereas deletion of any two yields full derepression. These elements correspond to a binding site for the abundant DNA-binding protein RAP1, an autonomous replicating sequence (ARS), and an as yet undistinguished region. From detailed deletion analysis of the E site we conclude that the ARS element contributes to silencer function in a capacity distinct from its role as an initiator of DNA replication. In addition, we find that strains deleted for any one of these elements comprise two genetically identical but phenotypically distinct types of cells: Those with HML apparently fully derepressed, and those with HML apparently completely repressed. These results reinforce the notion that epigenetic inheritance is an intrinsic characteristic of silencer action.