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In this paper I use clinical theory and illustration to explore details of the formulation of experience, which depends upon the metamorphosis of experience from not-me to feels-like-me. I take the position that the movement from not-me to feels-like-me, with the accompanying possibilities for formulating new meaning that open at such moments, happens when we not only know or feel something, but also, and simultaneously, sense ourselves in the midst of this process-that is, when we know and feel that it is we who are doing the knowing and feeling. When these two events co-occur, which depends upon the process of witnessing and the breach of dissociation, we come into possession of ourselves. Witnessing of one person by another is a process of recognition, but it is also a kind of affirmation performed by "someone who is trusted and justifies the trust and meets the dependence" (Winnicott 1971, p. 60).

We are used to the idea that trauma in the past interrupts our capacity to grasp the present. But present or recent trauma can have a similar dissociative effect on our capacity to experience the more distant past. Contemporary trauma can rob the past of its goodness, leaving one feeling as if the past is gone, dead, separated from the present. The vitalization of the present by the past or the past by the present requires that experiences be linked across time. These links are created, in both directions, via categories of experience characterized by shared affect (Modell 1990, 2006). Such categories are created, in turn, by metaphor; and the construction of these metaphors across time requires that one be able to occupy self-states in both the past and the present that can then bear witness to one another. Trauma can result in the dissociation of these self-states from one another, leading to a disconnection of present and past.

Even in the absence of others, we learn about ourselves by imaginatively listening to our own thoughts through the ears of the other. At the beginning of life, we need a witness to become a self. Later, patients listen to themselves as they imagine their analysts hear them, and in this way create new narrative freedom. The resolution of enactments is crucial in psychoanalytic treatment, not only because it expands the boundaries of the self, but also because it reinstitutes and broadens the range within which patient and analyst can witness one another's experience. Narrative is not the outcome of the analyst's objective interpretations, but an emergent, co-constructed, unbidden outcome of clinical process.

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

3'-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3' termini by processing yet are not polyadenylated, we asked whether 3' end maturation plays a role in chloroplast translation. A general characteristic of the 3' untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3'-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3' IR in strain Delta26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase beta-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in Delta26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3' end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase beta-subunit which accumulates in Delta26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3' end were not polysome associated. These results suggest that 3'-end processing of chloroplast mRNA is required for or strongly stimulates its translation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

A detailed analysis of RNA-protein complex formation in the 3' untranslated region of spinach chloroplast petD mRNA has been carried out. Five chloroplast proteins that interact with petD RNA in this region, which contains an inverted repeat sequence capable of forming a hairpin structure, have been identified. A 33-kDa protein recognizes specifically the double-stranded stem of the hairpin structure; mutations that disrupt base pairing at the base of the stem reduce or eliminate protein binding. A 57-kDa protein recognizes specifically an AU-rich sequence motif that is highly conserved in petD genes of different higher plant species. The 57-kDa protein and possibly the 33-kDa protein form stable complexes with petD RNA in vitro and may interact with each other. In addition, their interaction with petD RNA is highly sensitive to heparin. The three other proteins, of 100, 32, and 28 kDa, display little sequence or structural binding specificity apart from their preference for uridine-rich sequences. They also interact with the 3' untranslated regions of other chloroplast RNAs such as those of psbA and rbcL. The functions of these proteins in the regulation of petD gene expression, including possible roles in transcription termination and RNA stability, are discussed.