The v-y latissimus dorsi musculocutaneous flap in the reconstruction of large posterior chest wall defects.

Posterior chest wall defects are frequently encountered after excision of tumors as a result of trauma or in the setting of wound dehiscence after spine surgery. Various pedicled fasciocutaneous and musculocutaneous flaps have been described for the coverage of these wounds. The advent of perforator flaps has allowed the preservation of muscle function but their bulk is limited. Musculocutaneous flaps remain widely employed. The trapezius and the latissimus dorsi (LD) flaps have been used extensively for upper and middle posterior chest wounds, respectively. Their bulk allows for obliteration of the dead space in deep wounds. The average width of the LD skin paddle is limited to 10-12 cm if closure of the donor site is expected without skin grafting. In 2001 a modification of the skin paddle design was introduced in order to allow large flaps to be raised without requiring grafts or flaps for donor site closure. This V-Y pattern allows coverage of large anterior chest defects after mastectomy. We have modified this flap to allow its use for posterior chest wall defects. We describe the flap design, its indications, and its limitations with three clinical cases. Level of Evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266 .

Differential distribution of two microtubule-associated proteins, MAP2 and MAP5, during chick dorsal root ganglion development in situ and in culture.

Microtubule-associated proteins (MAPs) are essential components necessary for the early growth process of axons and dendrites, and for the structural organization within cells. Both MAP2 and MAP5 are involved in these events, MAP2 occupying a role predominantly in dendrites, and MAP5 being involved in both axonal and dendritic growth. In the chick dorsal root ganglia, pseudo-unipolar sensory neurons have a T-shaped axon and are devoid of any dendrites. Therefore, they offer an ideal model to study the differential expression of MAPs during DRG development, specifically during axonal growth. In this study we have analyzed the expression and localization of MAP2 and MAP5 isoforms during chick dorsal root ganglia development in vivo, and in cell culture. In DRG, both MAPs appeared as early as E5. MAP2 consists of the 3 isoforms MAP2a, b and c. On blots, no MAP2a could be found at any stage. MAP2b increased between E6 and E10 and thereafter diminished slowly in concentration, while MAP2c was found between stages E6 and E10 in DRG. By immunocytochemistry, MAP2 isoforms were mainly located in the neuronal perikarya and in the proximal portion of axons, but could not be localized to distal axonal segments, nor in sciatic nerve at any developmental stage. On blots, MAP5 was present in two isoforms, MAP5a and MAP5b. The concentration of MAP5a was highest at E6 and then decreased to a low level at E18. In contrast, MAP5b increased between E6 and E10, and rapidly decreased after E14. Only MAP5a was present in sciatic nerve up to E14. Immunocytochemistry revealed that MAP5 was localized mainly in axons, although neuronal perikarya exhibited a faint immunostaining. Strong staining of axons was observed between E10 and E14, at a time coincidental to a period of intense axonal outgrowth. After E14 immunolabeling of MAP5 decreased abruptly. In DRG culture, MAP2 was found exclusively in the neuronal perikarya and the most proximal neurite segment. In contrast, MAP5 was detected in the neuronal cell bodies and all along their neurites. In conclusion, MAP2 seems involved in the early establishment of the cytoarchitecture of cell bodies and the proximal axon segment of somatosensory neurons, while MAP5 is clearly related to axonal growth.

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteins.

Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro.

Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins.

The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.

Regulatory T cell defects are associated with autoimmunity in Wiskott-Aldrich Syndrome protein deficient mice

Abstract : The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive human primary immunodeficiency. It is caused by mutations in the gene encoding the hermatopoietic specific regulator of the actin cytoskeleton Wiskott-Aldrich Syndrome Protein (WASP). Importantly, a majority of affected patients develop autoimmunity including an inflammatory bowel disease (IBD)-like disease. WASP deficient mice share many similarities with the human WAS. One of these similarities is the spontaneous development of colitis. I have focused my dissertation studies on the pathogenesis of colitis in WASP deficient mice. Prior work from our laboratory had shown that lymphocytes were required and that CD4+ T cells sufficient for colitis development. This colitis was associated with a predominant Th2-cytokine skewing. I have contributed in exploring whether the Th2 cytokine IL-4 plays a role in disease maintenance. Using two approaches to neutralize IL-4, we found that this cytokine plays a role in disease maintenance. Natural CD4*CD25*Foxp3* regulatory T cells (nTreg cells) have been implicated in the pathogenesis of several autoimmune disorders. We found that WASP deficient mice have reduced nTreg cell numbers in peripheral lymphoid organs. This was associated with functional defects in suppressing T cell proliferation and preventing colitis induced by transfer of naïve T cells into SCID recipient, which lack lymphocytes. WASP deficiency affected homing of nTreg cells to lymphoid compartments, IL-2-mediated activation and secretion of the immunomodulatory cytokine IL-10. Finally, we could prevent colitis onset via adoptive transfer of WT nTreg cells prior to colitis development. This suggests that nTreg cells dysfunction is one of the mechanisms underlying colitis development in WASP deficient mice. Future directions will aim at deciphering the role of other immune cell types, the bacterial flora, and various cytokines in colitis development in this murine model of colitis. In addition, we believe that colitis in WASP deficient mice could serve as a useful tool to evaluate nTreg cells manipulation as novel therapeutic approach for IBD.

Regulation of HCF-1 function via proteolytic maturation and HCF-1 subunit interaction

Abstract : Host-Cell Factor 1 (HCF-1) was first discovered in the study of the herpes simplex virus (HSV) infection. HCF-1 is one of the two cellular proteins that compose the VP16-induced complex, a key activator of HSV lytic infection. lncleed, when HSV infects human cells, it is able to enter two modes of infection: lytic or latent. The V`P16-induced complex promotes the lytic mode and in so doing the virus targets important cellular regulatory proteins, such as HCF-1, to manipulate the status of the infected cell. Indeed, HCF-1 regulates human cell proliferation and the cell cycle at different steps. In human, HCF-1 is unusual in that it undergoes a process of proteolytic maturation that results from cleavages at six centrally located 26 amino acid repeats called HCF-1pro repeats. This generates a heterodimeric complex of stably associated amino- (HCF-1n) and carboxy- (HCF-1c) terminal subunits. The absence of the HCF-1 N or HCF-1; subunit leads predominantly to either G1 or M phase defects, respectively. We have hypothesized that HCF-1 forms a heterodimeric complex to permit communication between the two subunits of HCF-1 involved in regulating different phases of the cell cycle. Indeed, there is evidence for such inter-subunit communication because a point mutation called P134S in the HCF-1N subunit in the temperature-sensitive hamster cell line tsBN67 causes, addition to G1- phase defects associated with the HCF-1n subunit, M-phase defects similar to the defects seen upon loss of HCF-1 function. Furthermore, inhibition of the proteolytic maturation of HCF-1 by deletion of the six HCF-1pro repeats (HCF-1Aimo) also leads to M-phase defects, specifically cytokinesis defects leading to binucleation, indicating that there is loss of HCF-15 function in the absence of HCF-1 maturation. I demonstrate that individual point mutations in each of the six HCF-1pro repeats that prevent HCF-1 proteolytic maturation also lead to binucleation; however, this defect can be latgely rescued by the presence of just one HCF-1pRO sequence in I-ICF»1. These results argue that processing itself is important for the HCF-1g function. In fact, until now, the hypothesis was that the proteolytic processing per se is more important for HCF-1C function than the proteolytic processing region. But I show that processing per se is not sufticient to rescue multinucleation, but that the HCF-lpm sequence itself is crucial. This discovery leads to the conclusion that the I-ICF-1pRO repeats have an additional function important for HCF-le function. From the studies of others, one potential function of the HCF-lrxo tepeats is as a binding site for O-link NAcetyl glycosamine tansferase (OGT) to glycosylate an HCF-1n-sunbunit region called the Basic region. This new function suggests the Basic region of HCF-1n is also implicated in the communication between the two subunits. This inter-subunit communication was analyzed in more detail with the studies of the Pl34S mutation and the residues 382-450 region of HCF-l that when removed prevents HCF-l subunit association. I demonstrate that the point mutation also leads to a binucleation defect in Hela cells as well as in the tsBN67 cells. In addition, the effect of this mutation on the regulation of HCF-1c activity seems to interfere with that of the HCF-lpgg repeats because the sum of the deletion of the proteolytic processing region and the point mutation surprisingly leads to re-establishment of correct cytokinesis. The study of the 382-450 HCF-lN region also yielded surprising results. This region important for the association of the two subunits is also important for both HCF-1c function in M phase and G1 phase progression. Thus, I have discovered two main functions of this region: its role in the regulation of HCF-lc function in M phase and its involvement in the regulation of G1/S phase ?- an HCF-1n function. These results support the importance of inter-subunit communication in HCF-1 functions. My research illuminates the understanding of the interaction of the two subunits by showing that the whole HCF-1n subunit is involved in the inter-subunit communication in order to regulate HCF-1c function. For this work, I was concentrated on the study of cytokinesis; the first phenotype showing the role of HCF-1c in the M phase. Then, I extended the study of the M phase with analysis of steps earlier to cytokinesis. Because some defects in the chromosome segregation was already described in the absence of HCF-1, I decided to continue the study of M phase by checking effects on the chromosome segregation. I showed that the HCF-1n subunit and HCF-1pro repeats are both important for this key step of M phase. I show that the binucleation phenotype resulting from deletion or mutation in HCF-1pro repeats, Pl34S point mutation or the lack of the region 382-450 are correlated with micronuclei, and chromosome segregation and alignment defects. This suggests that HCF«lç already regulates M phase during an early step and could be involved in the complex regulation of chromosome segregation. Because one of the major roles of HCF-1 is to be a transcription regulator, I also checked the capacity of HCF-1 to bind to the chromatin in my different cell lines. All my recombinant proteins can bind the chromatin, except for, as previously described, the HCF-1 with the P134S point mutation, This suggests that the binding of HCF-1 to the chromatin is not dependant to the Basic and proteolytic regions but more to the Kelch domain. Thus, if the function of HCF-ig in M phase is dependant to its chromatin association, the intercommunication and the proteolytic region are not involved in the ability to bind to the chromatin but more to bind to the right place of the chromatin or to be associated with the co-factors. Résumé : L'étude de l'infection par le virus Herpes Simplex (HSV) a permis la découverte de la protéine HCF-1 (Host-Cell Factor). HCF-1 est une des protéines cellulaires qui font partie du complexe induit par VP16 ; ce complexe est la clef pour l'activation de la phase lytique de HSV. Afin de manipuler les cellules infectées, le complexe induit pas le VPIG devrait donc cibler les protéines importantes pour la régulation cellulaire, telles que la protéine HCF-1. Cette dernière s'avère donc être un senseur pour la cellule et devrait également jouer un rôle de régulation lors des différentes phases du cycle cellulaire. Chez l'humain, HCF-1 a la particularité de devoir passer par une phase de maturation pour devenir active. Lors de cette maturation, la protéine subit une coupure protéolytique au niveau de six répétitions composées de 26 acides aminés, appelé HCF-1pro repeats. Cette coupure engendre la formation d'un complexe formé de deux sous-unités, HCF-1n et HCF-1c, associées l'une à l'autre de façon stable. Enlever la sous-unité HCF-IN ou C entraîne respectivement des défauts dans la phase G1 et M. Nous pensons donc que HCF-1 forme un complexe hétérodimérique afin de permettre la communication entre les molécules impliquées dans la régulation des différentes phases du cycle cellulaire. Cette hypothèse est déduite suite à deux études: l'une réalisée sur la lignée cellulaire tsBN67 et l'autre portant sur l'inhibition de la maturation protéolytique. La lignée cellulaire tsBN67, sensible à la température, porte la mutation Pl 345 dans la sous-unité HCF-1n. Cette mutation, en plus d'occasionner des défauts dans la phase G1 (défauts liés à la sous-unité HCF-1N), a aussi pour conséquence d'entrainer des défauts dans la phase M, défauts similaires à ceux dus a la perte de la sous-unité HCF-1c. Quant à la maturation protéolytique, l'absence de la région de la protéolyse provoque la binucléation, défaut lié à la cytokinèse, indiquant la perte de la fonction de la sous-unité HCF-1c. Au cours de ma thèse, j'ai démontré que des mutations dans les HCF-1=no repeats, qui bloquent la protéolyse, engendrent la binucléation ; cependant ce défaut peut être corrigé pas l'ajout d'un HCF-1pro repeat dans un HCF-1 ne contenant pas la région protéolytique. Ces résultats soutiennent l'idée que la région protéolytique est importante pour le bon fonctionnement de HCF-1c. En réalité jusqu'a maintenant on supposait que le mécanisme de coupure était plus important que la région impliquée pour la régulation de la fonction de HCF-1;. Mais mon étude montre que la protéolyse n'est pas suffisante pour éviter la binucléation ; en effet, les HCF-1pro repeats semblent jouer le rôle essentiel dans le cycle cellulaire. Cette découverte conduit à la conclusion que les HCF-1pro repeats ont sûrement une fonction autre qui serait cruciale pour la foncton de HCF-1c. Une des fonctions possibles est d'être le site de liaison de l'O-linked N-acetylglucosamine transférase (OGT) qui glycosylerait la région Basique de HCF-1n. Cette nouvelle fonction suggère que la région Basique est aussi impliquée dans la communication entre les deux sous- unités. L'intercommunication entre les deux sous-unités ai été d'ailleurs analysée plus en détail dans mon travail à travers l'étude de la mutation Pl34S et de la région 382-450, essentielle pour l'association des deux sous»unités. J'ai ainsi démontré que la mutation P134S entraînait aussi des défauts dans la cytokinése dans la lignée cellulaire Hela, de plus, son influence sur HCF-1c semble interférer avec celle de la région protéolytique. En effet, la superposition de ces deux modifications dans HCF-1 conduit au rétablissement d'une cytokinése correcte. Concernant la région 382 à 450, les résultats ont été assez surprenants, la perte de cette région provoque l'arrêt du cycle en G1 et la binucléation, ce qui tend à prouver son importance pour le bon fonctionnement de HCF-1n et de HCF-1c. Cette découverte appuie par conséquent l'hypotl1èse d'une intercommunicatzion entre les deux sous-unités mettant en jeu les différentes régions de HCF-1n. Grâce à mes recherches, j'ai pu améliorer la compréhension de l'interaction des deux sous-unités de HCF-1 en montrant que toutes les régions de HCF-1n sont engagées dans un processus d'intercommunication, dont le but est de réguler l'action de HCF-1c. J'ai également mis en évidence une nouvelle étape de la maturation de HCF-1 qui représente une phase importante pour l'activation de la fonction de HCF-1c. Afin de mettre à jour cette découverte, je me suis concentrée sur l'étude de l'impact de ces régions au niveau de la cytokinése qui fut le premier phénotype démontrant le rôle de HCF-1c dans la phase M. A ce jour, nous savons que HCF-1c joue un rôle dans la cytokinèse, nous ne connaissons pas encore sa fonction précise. Dans le but de cerner plus précisément cette fonction, j'ai investigué des étapes ultérieures ai la cytokinèse. Des défauts dans la ségrégation des chromosomes avaient déjà été observés, ai donc continué l'étude en prouvant que HCF-1n et les HCF-1pro repeats sont aussi importants pour le bon fonctionnement de cette étape clef également régulée par HCF-1c. J' ai aussi montré que la région 382-450 et la mutation P134S sont associées à un taux élevé de micronoyaux, de défauts dans la ségrégation des chromosomes. L'une des fonctions principales de HCF-1 étant la régulation de la transcription, j'ai aussi contrôlé la capacité de HCF-1 à se lier à la chromatine après insertion de mutations ou délétions dans HCF-1n et dans la région protéolytique. Or, à l'exception des HCF-1 contenant la mutation P134S, la sous-unité HCF-1c des HCF-1 tronquées se lie correctement à la chromatine. Cette constatation suggère que la liaison entre HCF-1c et chromatine n'est pas dépendante de la région Basique ou Protéolytique mais peut-être vraisemblablement de la région Kelch. Donc si le rôle de HCF-1c est dépendant de sa capacité â activer la transcription, l'intercommunication entre les deux sous-unités et la région protéolytique joueraient un rôle important non pas dans son habileté à se lier à la chromatine, mais dans la capacité de HCF-1 à s'associer aux co-facteurs ou à se placer sur les bonnes régions du génome.

Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene.

In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon-intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows: NH2 - lipovitellin I - phosvitin (or phosvette II - phosvette I) - lipovitellin II - COOH.

Binding of d-methadone, l-methadone, and dl-methadone to proteins in plasma of healthy volunteers: role of the variants of alpha 1-acid glycoprotein.

The plasma concentrations of alpha 1-acid glycoprotein (AAG), albumin, triglycerides, cholesterol, and total proteins, as well as the plasma binding of racemic, d-methadone, and l-methadone were measured in 45 healthy subjects. The AAG phenotypes and the concentrations of AAG variants were also determined. The measured free fractions for racemic, d-methadone, and l-methadone were, respectively, 12.7% +/- 3.3%, 10.0% +/- 2.9%, and 14.2% +/- 3.2% (mean +/- SD). A significant correlation was obtained between the binding ratio (B/F) for dl-methadone and the total AAG concentration (r = 0.724; p less than 0.001). A multiple stepwise regression analysis showed that AAG was the main explanatory variable for the binding of the racemate. When concentrations of AAG variants were considered, a significant correlation was obtained between the binding ratio of dl-methadone and orosomucoid2 A concentration (r = 0.715; p less than 0.001), a weak correlation between dl-methadone and orosomucoid1 S concentration (r = 0.494; p less than 0.001), and no correlation between dl-methadone and orosomucoid1 F1 concentration (r = 0.049; not significant). Similar findings were obtained with the enantiomers. This study shows the importance of considering not only total AAG but also concentrations of AAG variants when measuring the binding of methadone and possibly of other drugs in plasma.

Multiple adenosine 3',5'-cyclic [corrected] monophosphate response element DNA-binding proteins generated by gene diversification and alternative exon splicing.

The protein sequence deduced from the open reading frame of a human placental cDNA encoding a cAMP-responsive enhancer (CRE)-binding protein (CREB-327) has structural features characteristic of several other transcriptional transactivator proteins including jun, fos, C/EBP, myc, and CRE-BP1. Results of Southwestern analysis of nuclear extracts from several different cell lines show that there are multiple CRE-binding proteins, which vary in size in cell lines derived from different tissues and animal species. To examine the molecular diversity of CREB-327 and related proteins at the nucleic acid level, we used labeled cDNAs from human placenta that encode two different CRE-binding proteins (CREB-327 and CRE-BP1) to probe Northern and Southern blots. Both probes hybridized to multiple fragments on Southern blots of genomic DNA from various species. Alternatively, when a human placental c-jun probe was hybridized to the same blot, a single fragment was detected in most cases, consistent with the intronless nature of the human c-jun gene. The CREB-327 probe hybridized to multiple mRNAs, derived from human placenta, ranging in size from 2-9 kilobases. In contrast, the CRE-BP1 probe identified a single 4-kilobase mRNA. Sequence analyses of several overlapping human genomic cosmid clones containing CREB-327 sequences in conjunction with polymerase chain reaction indicates that the CREB-327/341 cDNAs are composed of at least eight or nine exons, and analyses of human placental cDNAs provide direct evidence for at least one alternatively spliced exon. Analyses of mouse/hamster-human hybridoma DNAs by Southern blotting and polymerase chain reaction localizes the CREB-327/341 gene to human chromosome 2. The results indicate that there is a dichotomy of CREB-like proteins, those that are related by overall structure and DNA-binding specificity as well as those that are related by close similarities of primary sequences.