Christine B. Michalowski

A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant.

Seawater-strength salt stress of the ice plant (Mesembryanthemum crystallinum) initially results in wilting, but full turgor is restored within approximately 2 days. We are interested in a mechanistic explanation for this behavior and, as a requisite for in-depth biochemical studies, have begun to analyze gene expression changes in roots coincident with the onset of stress. cDNAs that suggested changes in mRNA amount under stress were found; their deduced amino acid sequences share homologies with proteins of the Mip (major intrinsic protein) gene family and potentially encode aquaporins. One transcript, MipB, was found only in root RNA, whereas two other transcripts, MipA and MipC, were detected in roots and leaves. Transcript levels of MipB were of low abundance. All transcripts declined initially during salt stress but later recovered to at least prestress level. The most drastic decline was in MipA and MipC transcripts. MipA mRNA distribution in roots detected by in situ hybridization indicated that the transcript was present in all cells in the root tip. In the expansion zone of the root where vascular bundles differentiate, MipA transcript amounts were most abundant in the endodermis. In older roots, which had undergone secondary growth, MipA was highly expressed in cell layers surrounding individual xylem strands. MipA was also localized in leaf vascular tissue and, in lower amounts, in mesophyll cells. Transcripts for MipB seemed to be present exclusively in the tip of the root, in a zone before and possibly coincident with the development of a vascular system. MipA- and MipB-encoded proteins expressed in Xenopus oocytes led to increased water permeability. mRNA fluctuations of the most highly expressed MipA and MipC coincided with turgor changes in leaves under stress. As the leaves regained turgor, transcript levels of these water channel proteins increased.

Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during Crassulacean acid metabolism induction in the common ice plant.

The common ice plant is a facultative halophyte in which Crassulacean acid metabolism, a metabolic adaptation to arid environments, can be induced by irrigating plants with high levels of NaCl or by drought. This stress-induced metabolic transition is accompanied by up to a 50-fold increase in the activity of phosphoenolpyruvate carboxylase (PEPCase). To analyze the molecular basis of this plant response to water stress, we have isolated and characterized two members of the PEPCase gene family from the common ice plant. The PEPCase isogenes, designated Ppc1 and Ppc2, have conserved intron-exon organizations, are 76.4% identical at the nucleotide sequence level within exons, and encode predicted polypeptides with 83% amino acid identity. Steady-state levels of mRNAs from the two genes differ dramatically when plants are salt-stressed. Transcripts of Ppc1 increase about 30-fold in leaves within 5 days of salt stress. In contrast, steady-state levels of Ppc2 transcripts decrease slightly in leaf tissue over the same stress period. Steady-state levels of transcripts of both genes decrease in roots over 5 days of salt stress. We have used in vitro transcription assays with nuclei isolated from leaves to demonstrate that the increased expression of Ppc1 caused by water stress occurs in part at the transcriptional level.

Nucleotide sequence of the cyanelle genome fromCyanophora paradoxa

The complete nucleotide sequence of the cyanelle genome ofCyanophora paradoxa Pringsheim strain LB 555 was determined (accession number U30821). The circular molecule is 135,599 base pairs in length. The physical map of this DNA molecule is shown along with identified genes and open reading frames.

Mesembryanthemum crystallinum, a higher plant model for the study of environmentally induced changes in gene expression

Plants have evolved several strategies to deal with water stress brought about by changes in soil water potential or solute concentration (Hanson & Hitz, 1982; Morgan, 1984). One solution to long-term periods of water stress is avoidance. This often involves rapid completion of ontogeny or prolonged periods of dormancy. Many halophytes adapt to changes in soil salinity by accumulating inorganic ions in the vacuole. The osmotic potential of the cytoplasm is balanced by the synthesis and accumulation of biologically compatible solutes such as proline, betaine, polyamines, sugars or sugar alcohols (Hanson & Hitz, 1982; Flores et al., 1985). A few species actively secrete or sequester salt via salt glands or salt hairs (Hill & Hill, 1976). Some facultative halophytes such as Mesembryanthemum crystallinum switch to crassulacean acid metabolism (CAM),

Induction of a ribosome-inactivating protein upon environmental stress.

Transcripts of altered abundance in RNA from unstressed and 500 mm salt-shocked Mesembryanthemum crystallinum (common ice plant) were detected by reverse-transcription differential display (RT-DD). One transcript, Rip1, was of very low abundance in unstressed plants and was strongly induced by stress. RNA blot hybridizations showed strong induction and a diurnal rhythm of transcript abundance with a maximum each day around the middle of the light phase. Rip1 encodes a reading frame of 289 amino acids (molecular mass 32652), RIP1, with homology to single-chain ribosome inactivating proteins (rRNA N-glycosidases). The deduced amino acid sequence is 31.7% identical to pokeweed antiviral protein RIP-C (overall similarity 66.5%) with highest identity in domains of documented functional importance. RT-DD also detected mRNA for pyruvate,orthophosphate dikinase (PPDK) which has already been shown to be stress-induced in the ice plant [16]. RIP1, expressed in Escherichia coli, showed rRNA N-glycosidase activity against ice plant and rabbit reticulocyte ribosomes. The induction of Rip1 coincides with the transition period during which global changes in translation lead to adaptation of the ice plant to salt stress.

RecA-dependent cleavage of LexA dimers.

A critical step in the SOS response of Escherichia coli is the specific proteolytic cleavage of the LexA repressor. This reaction is catalyzed by an activated form of RecA, acting as a co-protease to stimulate the self-cleavage activity of LexA. This process has been reexamined in light of evidence that LexA is dimeric at physiological concentrations. We found that RecA-dependent cleavage was robust under conditions in which LexA is largely dimeric and conclude that LexA dimers are cleavable. We also found that LexA dimers dissociate slowly. Furthermore, our evidence suggests that interactions between the two subunits of a LexA dimer can influence the rate of cleavage. Finally, our evidence suggests that RecA stimulates the transition of LexA from its noncleavable to its cleavable conformation and therefore operates, at least in part, by an allosteric mechanism.

Cyanelle DNA from Cyanophora paradoxa

SummaryCyanelles which have been found in few eukaryotic organisms are photosynthetically active organelles which strikingly resemble cyanobacteria. The complexity of the cyanelle genome in Cyanophora paradoxa (127 Kbp) is too low to consider them as independent organisms in a symbiotic relationship. In order to correlate cyanelle genome and gene structure with those of plastid chromosomes of other plants, a circular map of the cyanelle DNA from Cyanophora paradoxa (strain LB555 UTEX) has been constructed using the restriction endonucleases SalI (generating 6 DNA fragments), BamHI (6), SalI (5), XhoI (9), and BglII (19).Besides the rRNA genes (16S, 23S, 5S), genes for 14 proteins have been located on this circular map. Among those are components of several multienzyme complexes involved in photosynthetic electron transport, as well as the large subunit of ribulose-1,5-bisphosphate carboxylase and two ribosomal proteins. All the probes used, were derived from a collection of spinach chloroplast DNA clones. Hybridization experiments showed signals to DNA fragments primarily from the large single-copy region of cyanelle DNA. The arrangement of genes on cyanelle DNA is different from that on spinach chloroplast DNA. However, genes which have been shown to be cotranscribed in spinach chloroplasts are also clustered on cyanelle DNA.

Sequence Tolerance of the Phage λ PRM Promoter: Implications for Evolution of Gene Regulatory Circuitry

Complex gene regulatory circuits can have a large number of interlocking components. This degree of interconnectivity raises two issues. First, how did these circuits evolve? Second, how can we understand the behavior of these existing circuits and predict their behavior in the face of small changes in parameters such as promoter strength? For these and other reasons, we have been analyzing the behavior of the regulatory circuitry of phage λ in the intact system. This system is probably the best-understood complex circuit (38, 39). Most, if not all, of the regulatory interactions have been identified, and most of these are well characterized at the mechanistic level. Previous analysis of this circuit generally has been carried out in uncoupled systems (such as the use of reporter genes and fusions with the lac promoter), an approach necessary to disentangle the causality of this system. With a circuit diagram in hand, it is now possible to return to the intact circuit and ask how particular changes affect the overall operation of the system. Many of the critical interactions in the λ circuit center on a complex regulatory region termed the OR region (Fig. ​(Fig.1C).1C). This ∼100-bp region is densely packed with cis-acting sites, including two promoters and three sites to which both the CI and Cro repressors can bind (38). In addition, the promoters and repressor-binding sites overlap extensively. CI and Cro regulate the expression of these promoters, and their actions determine the overall behavior of the regulatory circuitry. FIG. 1. Structure of the λJL387 vector and sequence of PRM. All maps are to scale, as indicated. (A) Comparison with l+. The indicated BsrGI and XhoI sites in λJL351 were introduced; that for BsrGI is the only BsrGI site to the right of ... The λ circuitry is bistable—that is, lambda can exist in either of two stable epigenetic states, the lytic state and the lysogenic state. A choice is made between these two states soon after infection; this choice involves regulatory elements (CII, CIII, and PRE [see reference 14]) that we do not consider here. Once a particular choice is made, it is stabilized by the interactions of CI or Cro with the OR region. In the lytic state, Cro is made from PR. At moderate concentrations, Cro binds preferentially to OR3, repressing PRM but not affecting its own expression; at higher levels, Cro also binds to OR1 and OR2, partially repressing its own synthesis. Hence, if Cro but not CI is present, this pattern is perpetuated. In the lysogenic state, by contrast, CI but no Cro is present; CI binds tightly to OR1 and cooperatively to OR2 but only weakly to OR3. Binding to OR1 and/or OR2 represses PR. CI bound to OR2 also stimulates expression of PRM about 10-fold (31); hence, CI acts in this context as an activator of its own expression in a positive feedback loop. Again, if CI but not Cro is present, this pattern is perpetuated by the behavior of the circuitry. At higher CI levels, PRM is partially repressed by binding of CI to OR3, both directly and by a cooperative long-range interaction with CI molecules bound at a distant regulatory region termed OL (7, 8, 40). It is of interest to ask how this particular arrangement and spacing of cis-acting sites benefits the phage, how it arose during the course of evolution, and how the sequences of the sites were refined during the course of evolution. We chose to address the last question by asking whether one of these cis-acting sites, the PRM promoter, could tolerate substantial genetic changes and still allow relatively normal behavior of the intact circuitry. Although unstimulated PRM is a relatively weak promoter, it differs from the consensus σ70-specific promoter in only 4 positions out of 12 (Fig. ​(Fig.1D).1D). Is its resemblance to the consensus important, or could it tolerate substantial changes and still preserve its function? Previous work and our unpublished studies have identified several point mutations in PRM that weaken or strengthen it, and these mutations affect the behavior of the λ circuitry (12, 31, 42, 43; our unpublished work), suggesting that its sequence is important. It is unclear whether more extensive changes in the promoter would destroy the operation of the circuitry. For instance, promoter mutations might also affect other processes operating in this complicated regulatory region. In this work, we have tested the effects of extensive changes in PRM on the operation of the λ circuitry. In order to facilitate this and similar studies, it would be useful to be able to clone variants of the OR region into a wild-type background. This would allow one, for example, to use intensive site- or region-directed mutagenesis to create large pools of recombinant molecules and then to introduce these into intact genomes and assess their effects by using the powerful screens and selections afforded by λ genetics. This approach could be extended to combinatorial approaches in which many combinations are made and tested simultaneously. We describe here the isolation of a cloning vector that allows such manipulations in the OR regulatory region and use it to carry out extensive mutagenesis of PRM. We find that sequences differing markedly from the wild-type sequence can confer nearly normal behavior on the λ circuitry.

The cyanelle S10 spc ribosomal protein gene operon from Cyanophora paradoxa

SummaryIn Cyanophora paradoxa photosynthetic organelles termed cyanelles perform the functions of chloroplasts in higher plants, while the structural and biochemical characteristics of the cyanelle are essentially cyanobacterial. Our interest in studying the evolutionary relationship between cyanelles and chloroplasts led us to focus on cyanelle-encoded genes of the translational apparatus, specifically genes equivalent to those of the bacterial S10 and spc operons. The structure of a large ribosomal protein gene cluster from cyanelle DNA was characterized and compared with that from plastids and bacteria. Sequences of the following cyanelle genes encompassing 4.8 kb are reported here: 5′-rpl22-rps3-rpl16-rps17-rpl14-rpl5-rps8-rpl6-rpl18-rps5-3′. Cyanelles contain five more ribosomal protein genes than do higher plant chloroplasts and four more genes than Euglena gracilis plastids in the S10/spc region of this gene cluster. The gene encoding rpl36 is absent, in contrast to the case in other plastid DNAs. These genes, including the previously characterized genes rpl3, rpl2 and rps19, are transcribed as a primary transcript of ∼7500 nucleotides. The occurrence of transcripts smaller than this presumptive primary transcript suggests that it is processed into defined segments. Transcription terminates 3′ of rps5 where a 40 by hairpin with one mismatch (−42.2 kcal) may be folded. Immediately downstream of rps5 an open reading frame, ORF492, is contained on a separate transcript. A comparison of gene content, operon structure and deduced amino acid sequence of the genes in the S10 and spc operons from different organisms supports the notion that cyanelles are intermediary between known plastids and cyanobacteria.

Positive Autoregulation of cI Is a Dispensable Feature of the Phage λ Gene Regulatory Circuitry

Complex gene regulatory circuits contain many features that are likely to contribute to their operation. It is unclear, however, whether all these features are necessary for proper circuit behavior or whether certain ones are refinements that make the circuit work better but are dispensable for qualitatively normal behavior. We have addressed this question using the phage λ regulatory circuit, which can persist in two stable states, the lytic state and the lysogenic state. In the lysogenic state, the CI repressor positively regulates its own expression by stimulating transcription from the PRM promoter. We tested whether this feature is an essential part of the regulatory circuitry. Several phages with a cI mutation preventing positive autoregulation and an up mutation in the PRM promoter showed near-normal behavior. We conclude that positive autoregulation is not necessary for proper operation of the λ circuitry and speculate that it serves a partially redundant function of stabilizing a bistable circuit, a form of redundancy we term “circuit-level redundancy.” We discuss our findings in the context of a two-stage model for evolution and elaboration of regulatory circuits from simpler to more complex forms.