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Presents a list of articles for publication in future issues of the "European Journal of Biochemistry". Conformation of physalaemin; Association of cross-linked proteins with a specific mRNA in the cytoplasm of HeLa cells; Transport of proteins into chloroplasts.

This article presents a list of some of the forthcoming papers in biochemistry to be published in this journal. Some of the papers listed are "The Organization of the Chloroplast DNA in wheat and maize in the region containing the LS Gene," by B. Koller, H. Delius and T.A. Dyer, "Effect of Carbamination on the Buffering Power of Purified Human Hamoglobins A Solutions At Two Temperatures," by M. Castaing, E. Bursaux and C. Poyart, "The Complete Nucleotide of the Chicken Ovotransferrin mRNA," by J.M. Jeltsch and P. Chambon, "The Primary Structure of Hen Ovotransferrin," by J. Williams, T.C. Elleman, I.B. Kington, A.G. Wilkins and K.A. Kuhn.

In this paper the mode of action of IF-1 in 40-S initiation complex formation was studied with MS 2 RNA as messenger. Using initiation factors IF-2 and IF-3 labeled in vitro it appeared that IF-I did not influence the binding of these factors in the absence of fMet-tRNA. However, in the presence of fMet-tRNA it was found that the enhancement of the fMet-tRNA binding by IF-1 was accompanied with an equimolar increase in binding of IF-2. Moreover, it appeared that also in absence of IF-I, fMet-tRNA binding is coupled with an equimolar enhancement of the IF-2 binding, which suggests the existence of a preribosomal complex between IF-2 and fMet-tRNA. The apparent K,, values for both the binding of fMet-tRNA and IF-2 to 30-S subunits were determined and appeared to be equal, which makes a functioning of such a preribosomal complex in protein initiation very likely. The participation of GTP in this complex will be discussed. Functions of IF-I in dissociation and recycling of IF-2, described by others, and the stimulation on the 30-S subunit level might well be explained as pleiotropic effects of one basic action of IF-1, i.e. a conformational change of 30-S subunits. [ABSTRACT FROM AUTHOR]

Transcriptional gene silencing has broad applications for studying gene function in planta. In maize, a large number of genes have been identified as tassel-preferred in their expression pattern, both by traditional genetic methods and by recent high-throughput expression profiling platforms. Approaches using RNA suppression may provide a rapid alternative means to identify genes directly related to pollen development in maize. The male fertility gene Ms45 and several anther-expressed genes of unknown function were used to evaluate the efficacy of generating male-sterile plants by transcriptional gene silencing. A high frequency of male-sterile plants was obtained by constitutively expressing inverted repeats (IR) of the Ms45 promoter. These sterile plants lacked MS45 mRNA due to transcriptional inactivity of the target promoter. Moreover, fertility was restored to these promoter IR-containing plants by expressing the Ms45 coding region using heterologous promoters. Transcriptional silencing of other anther-expressed genes also significantly affected male fertility phenotypes and led to increased methylation of the target promoter DNA sequences. These studies provide evidence of disruption of gene activity in monocots by RNA interference constructs directed against either native or transformed promoter regions. This approach not only enables the correlation of monocot anther-expressed genes with functions that are important for reproduction in maize, but may also provide a tool for studying gene function and identifying regulatory components unique to transcriptional gene control. [ABSTRACT FROM AUTHOR]

RNA polymerase II (pot II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homotogue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β-propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pot II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination. [ABSTRACT FROM AUTHOR]

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm 10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization. [ABSTRACT FROM AUTHOR]

Gel filtration of Peking duck eye lens proteins reveals a component eluting just behind δ-crystallin and comprising approximately 10% of the total soluble protein. The native Mr of this additional component is estimated to be 120000; it appears to be composed of three identical chains of Mr 38000 and pI 7.5. Circular dichroic spectroscopy showed a relatively high α-helical content. No immunological cross-reactivity is found with α-, β,- γ- or δ-crystallins, and partial amino acid sequence determinations likewise failed to reveal any similarity with other known crystallins. We conclude that this protein represents another and novel family of eye lens proteins, for which we propose the designation ε-crystallin. ε-Crystallin is translated from a 1450-base mRNA, which has been partially purified. ε-Crystallin is found scattered among avian and reptilian taxa, but not in other vertebrates. Its rate of evolutionary change seems to be as slow as that of α- and β-crystallins. [ABSTRACT FROM AUTHOR]

We have recently identified an antisense RNA (RNAα) that regulates the expression of the fatA iron transport gene encoding the outer membrane receptor for the iron--anguibactin complex. In this work, we demonstrate that RNAα also inhibits the expression of fatB, which encodes a 35 kDa iron transport protein and has domains homologous to other periplasmic transport proteins. The expression of fatA and fatB is repressed under iron-rich conditions, in which RNAα is induced. RNAα is homologous to two-thirds of the coding region of fatB. By cloning RNAα coding sequences immediately downstream of a tet promoter, we were able to obtain constitutive expression of the antisense RNA. The cloned region contains approximately 83% of the 650 nucleotide RNAα and is complementary to only 51% of the fatB mRNA but is still capable of causing a repression of the expression of the fatB gene. Our results in this work demonstrate that RNAα probably affects the stability of the fatB-specific mRNA. [ABSTRACT FROM AUTHOR]

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S1S binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation. [ABSTRACT FROM AUTHOR]

The chromophoric protein phycocyanin is the major protein in the phycobilisome rod of the cyanobacterium Synechococcus 6301 (formerly designated Anacystis nidulans sp. UTEX 625). The gene clusters coding for the β- and α-subunits of phycocyanin are duplicated on the chromosome of Synechococcus 6301 and separated by an intergenic region 2.5 kb long. The structure of the phycocyanin operons of the phycobilisome mutant strains AN112 and AN135 was compared to that of wild-type Synechococcus 6301. Both mutants have an altered phycobilisome structure resulting in the appearance of rods of a shorter overall length. Genetic mapping indicated that the mutant strain AN112 completely lacked the intergenic region and carried only one set of phycocyanin subunit genes. No gross structural difference in the genetic organization of the corresponding region of mutant strain AN135 was detected. Northern blot analysis and primer extension experiments were used to monitor the transcriptional pattern of the phycocyanin rod operon. It was found that AN112 had lost the 3.7kb minor mRNA, which covers the intergenic region, and only produced two major 1.3 and 1.4 kb mRNA species. These transcripts were identified as fusion products of the 5′ end of the transcriptional unit originating from the promoter region of the left-hand phycocyanin gene cluster and the 3′ end of the transcriptional unit covering the right-hand phycocyanin gene cluster. The mutant strain AN135 had a transcriptional pattern very similar to that of the wild type. The level of transcription of the major transcripts covering the phycocyanin gene clusters was similar in the wild-type and mutant strains. The results indicate that genes coding for two of the linker proteins, namely the 30 and 33kD polypeptides, are located in the intergenic region between the duplicated phycocyanin gene clusters in Synechococcus 6301. The results also show that loss of the right-hand phycocyanin promoter does not drastically impair phycocyanin synthesis. [ABSTRACT FROM AUTHOR]

mRNA extracted from rat duodenum, kidney and cerebellum was translated in a cell-free reticulocyte lysate system in the presence of L-[35S]methionine. Vitamin-D-dependent calcium-binding proteins (D-CaBPs) were identified by immunoprecipitation using antibodies specific to duodenal D-CaBP (7500 Mr) and cerebellar D-CaBP (28000 Mr). When duodenal mRNA was translated, the immunoprecipitated polypeptide, obtained using antibodies to duodenal D-CaBP, comigrated with the pure small D-CaBP. Only the addition of unlabeled small duodenal D-CaBP prevented the immunoprecipitation of the major protein. Likewise, when mRNA extracted from the kidney and cerebellum was translated, the product immunoprecipitated by antibodies specific to large mammalian D-CaBP was electrophoretically similar to pure 28000-Mr protein, being displaced only by the addition of unlabeled large D-CaBP. The yield of the duodenal D-CaBP synthesized in the reticulocyte lysate assay was remarkably high (about 10%) compared to that of the large D-CaBP with renal (1%) or cerebellar (0.4%) mRNA. In the absence or presence of microsomal membranes, proteins of similar molecular weight were synthesized, suggesting that the biosynthesis of both large and small D-CaBPs do not involve the processing of leader sequences. Moreover in our experimental conditions duodenal poly(A)-rich RNA was unable to direct the synthesis of large D-CaBP while the mRNAs extracted from kidney and cerebellum did not code for the small D-CaBP. Our data indicate that two distinct mRNAs, coding for small and for large vitamin-D-dependent CaBPs, are expressed in specific tissues of the rat. [ABSTRACT FROM AUTHOR]

Combining messenger RNA from one kind of secretory cell with the cytoplasm of another such cell can reveal the nature and specificity of protein export mechanisms. We show that messenger RNAs from secretory cells of chickens, rats. mice, frogs, guinea-pigs, locusts and barley plants, when injected into Xenopus oocytes, direct the synthesis and export of proteins. Chicken ovalbumin, Xenopus albumin, mouse thyroid-stimulating hormone, locust vitellin and guinea-pig milk proteins were identified using specific antibodies, whilst chicken lysozyme and ovomucoid, rat albumin, Xenopus vitellogenin and rat seminal vesicle basic proteins were identified provisionally from their molecular weights. Certain endogenous proteins are sequestered and secreted although most oocyte proteins are not exported. Similarly the major polyoma viral protein and the simian virus 40 and polyoma tumour antigens are retained within the oocyte. Radioactive proteins exported by oocytes programmed with chicken oviduct or Xenopus liver RNA are not re-exported in detectable amounts when injected into fresh oocytes, nor is there secretion of chicken oviduct or guinea-pig mammary gland primary translation products prepared using wheat germ extracts. Thus the export of secretory proteins from oocytes cannot be explained by leakage and may require a cotranslational event. The secretory system of the oocyte is neither cell-type nor species-specific yet is highly selective. We suggest that the oocyte can be used as a general surrogate system for the study of gene expression, from transcription through translation to the final subcellular or extracellular destination of the processed protein. [ABSTRACT FROM AUTHOR]

cDNA synthesized on purified vitellogenin mRNA from Xenopus liver was hybridized to the template in formamide/urea at 22°C to avoid degradation of the RNA. The hybrids formed were visualized by spreading for electron microscopy. Contour length measurements proved that most of the RNA molecules in the hybrids were still intact showing the expected molecular weight of 2.3 × 106. The hybridized cDNA corresponded on the average to 12% of the RNA length. In about 80% of the molecules the cDNA was located at one end Since cDNA synthesis was primed by oligo(dT), the terminal duplex region marks the 3′ end of the vitellogenin mRNA molecule. Internal duplex regions were mainly located at a specific position starting about 2800 nucleotides from the 3′ end. Since the cDNA hybridizing at the internal position could specifically be synthesized on a vitellogenin RNA fragment isolated on poly(U)-Sepharose as an oligo(A)-containing RNA, we conclude that cDNA synthesis is not only initiated by the poly(A) of the 3′ end, but also by a specific internal sequence. [ABSTRACT FROM AUTHOR]

The most abundant poly(A)-containing RNA accounting for approximately half the total is regulated by testosterone in rat ventral prostate. A complementary DNA probe to these sequences was isolated by hybridizing the total cellular poly(A)-containing RNA with its complementary DNA to an rot½ value of 2 × 10-2 mol 1-1 s and separating the abundant cDNA from the non-abundant cDNA by hydroxyapatite chromatography. The abundant cDNA, complementary to about three different poly(A)-containing RNA sequences comprising 45% of the total poly(A)-containing RNA, has been used to investigate the effect of androgens on their metabolism. Following castration there is a progressive decrease in the abundant sequences from 450% of the total in normal animals to less than 0.03% after 14 days; testosterone administration in vivo results in their regeneration. The sequences were not detected in seminal vesicle, liver, heart or spleen but represented about 0.01% of the poly(A)-containing RNA of kidney. Translation of the poly(A)-containing RNA in vitro in a cell-free system derived from wheat germ resulted in the synthesis of four major proteins which could be separated by polyacrylamide gel electrophoresis. Their synthesis, as measured by [35S]methionine incorporation, accounted for 40–50% of the total incorporation and declined after castration but was restored by testosterone treatment. We conclude that a class of abundant poly(A)-containing RNA which codes for four major proteins is regulated by testosterone in rat ventral prostate. [ABSTRACT FROM AUTHOR]

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome. [ABSTRACT FROM AUTHOR]

The article focuses on a study of an RNA-editing event in the ADAR2 pre-mRNA. The auto-editing event in intron 4 creates a splice acceptor. The inclusion of an additional 47 nucleotides, generating a 1 frame shift that produces a truncated, catalytically inactive version of the protein. The alternative splicing by editing provides an autoregulatory feedback loop.