Non-synonymous gene polymorphisms in the secretory signal peptide of human TGF-β1 affect cellular synthesis but not secretion of TGF-β1

We have demonstrated that gene polymorphisms within the N-terminal leader sequence of TGF-β1 contribute to the outcome of hepatic fibrogenesis. In addition, the polymorphism at codon 25 affects TGF-β1 production in peripheral blood leukocytes. Therefore, it is general assumed that these polymorphisms influence cellular secretion of this cytokine. In the present study, we analysed if this widespread hypothesis is true. We cloned FLAG-tagged CMV-driven human full-length TGF-β1 expression constructs of the different allelic variations (i.e. 10Leu/25Arg, 10Pro/25Pro and 10Pro/25Arg) and transfected them into the immortal hepatic stellate cell line LX-2 and Chinese Hamster Ovary cells. Surprisingly, the allelic variants carrying a proline either in codon 10 or 25 showed overall reduced expression as assessed by Western blot and quantitative ELISA. We conclude that the allelic variations within the signal sequence influence the expression and not secretion of TGF-β1. Detailed RNA structure prediction analysis further suggests that the individual variants form different secondary structures.     

DNA sequence analysis of the rat RT1. B α gene

The major histocompatibility complex (MHC) of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1.B and RT1.D. The RT1. B gene was isolated for a Sprague-Dawley (RT1^b) rat genomic library using a rat RT1.B chain cDNA as a hybridization probe. The coding and the majority of the intron DNA sequence was determined. The structure of the RT1. B gene is equivalent to that of H-2 and HLA chain genes. Comparison of the nucleotide and predicted amino acid sequences of the RT1.B gene with those of the H-2 and HLA genes revealed a high degree of overall sequence conservation. However, two regions of the first external domain (l), residues 19–23 and 45–78, exhibit marked sequence variation. Two blocks of conserved nucleotide sequence were identified in the 5 promoter region of the RT1. B gene that have been described in all MHC class II genes sequenced to date. These conserved sequences may be involved in the coordinate regulation of expression of class II genes. The cloned RT1.B gene was efficiently transcribed when transfected to mouse L cells.     

DNA polymorphisms in the 5′-flanking region of the HLA-DQA1 gene

The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5 flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5 flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5 end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M72411. Address correspondence and offprint requests to: J. Guardiola.     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

The isolation of genomic recombinants for the human apolipoprotein B gene and the mapping of three common DNA polymorphisms of the gene —a useful marker for human chromosome 2

Summary We have used four independently isolated cDNA probes for human apolipoprotein B (apo B), to isolate overlapping genomic recombinants for the 3 portion of the apo B gene. The cDNA clones and a unique fragment from the genomic recombinant have been used to identify the human apo B gene in DNA from a series of roden x human somatic cell hybrids. Our results provide evidence for the assignment of this gene to the short arm of human chromosome 2 (p23-pter). We have used the cDNA probes to identify three common DNA polymorphisms. The first, detected with the restriction enzyme XbaI and our probe pAB4, has a rare allele frequency of 0.48. The other two polymorphisms are detected with the probe pAB3. The enzyme MspI detects at least three alleles, with frequencies of 0.67, 0.16 and 0.15, while that detected with the enzyme EcoRI has a rare allele frequency of 0.12. The relative position of these polymorphisms has been mapped using the genomic recombinants.Investigation of a small number of haplotypes indicares that there is linkage equilibrium between the polymorphisms, which have a total polymorphism information content (PIC) value of more than 0.8. These polymorphisms will provide useful markers for genetic studies on chromosome 2 and for the analysis of the involvement of variants of the apo B gene in the development of hyperlipidaemia.     

Expression of the Thomsen-Friedenreich antigen and of its putative carrier protein mucin 1 in the human placenta and in trophoblast cells in vitro

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an ‘uncloneable’ phenotype

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this ‘uncloneable’ fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNA^Thr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNA^Thr. These results imply that the HeLa mitochondrial tRNA^Thr gene is responsible for the ‘uncloneable’ phenotype of this region of human mitochondrial (mt) DNA.A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNA^Thr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T → G:C transitions. A preference for A:T → G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.     

Androgen dynamics in vitro in the human prostate gland. Effect of oestradiol-17β

Normal, hyperplastic and adenocarcinomatous human prostatic tissue was perfused in vitro with radioactively labelled androstenedione, testosterone and 5alpha-dihydrotestosterone with and without added oestradiol-17beta. Various parameters of tissue-steroid relationship were measured at the steady state. When oestradiol (0.11 or 0.22mumol/l) was added to the perfusing medium, the entry of the steroids into the tissue and their metabolism was increased in the majority of the glands studied. The ;uptake' of all the steroids varied, in response to the addition of oestradiol, in both normal and adenocarcinomatous glands in a way differing from the response of hyperplastic glands. As a consequence, the tissue clearance of the steroids, particularly of androstenedione and testosterone, increased in normal and adenocarcinomatous glands in the presence of oestradiol, and decreased in the hyperplastic tissues. At a concentration 0.33mumol/l, oestradiol decreased the entry of the steroids in all the tissues studied, while the clearance of steroids tended to decrease. The significance of these findings in terms of the regulation of androgen dynamics in vivo in the normal and diseased human prostate, with particular regard to the response to oestrogen treatment, is discussed.     

Ethylene and cytokinin interaction in the morphogenesis of Pelargonium × hortorum L.H. Bailey in vitro

Pelargonium × hortorum ‘Grand Prix’ which is susceptible to leaf yellowing and ‘Bergpalais’ which is not susceptible to leaf yellowing were chosen for the experiments. Ethylene production and action as well as the associated morphological response of Pelargonium shoots grown in the presence of a precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene inhibitors: α-aminooxyacetic acid (AOA) and silver nitrate (AgNO₃) and different cytokinins: (meta-topolin) (mT) or 6-benzylaminopurine (BAP) were studied. It was found that ‘Grand Prix’ was more sensitive to ethylene than ‘Bergpalais’ and it showed the leaf yellowing in response to 0.1 mg l^?1 ACC. Moreover, it was noted that ACC added separately or together with cytokinin influenced Pelargonium morphogenesis. Depending on the concentration of ACC (0.1–2.0 mg l^?1), it either stimulated or inhibited shoot and root formation as well as the growth of shoots and leaf blades. ACC-induced leaf yellowing in ‘Grand Prix’ was effectively inhibited by mT. In contrast, BAP did not enhance shoot quality. Simultaneously, the presence of mT in the medium resulted in up to a twofold increase in the ethylene production by ‘Grand Prix’ shoots throughout the culture period compared with the shoots growing on the BAP-medium. The inhibitor of ethylene action (AgNO₃) added with cytokinin prevented the yellowing of Pelargonium shoots, but simultaneously influenced the formation of mature shoots with limited long-term multiplication potential. The shoots of P. × hortorum ‘Grand Prix’ treated with AgNO₃ and mT emitted two- and sevenfold more ethylene after 11th and 21st day of culture compared with those treated with AgNO₃ and BAP. It is suggested that mT inhibits the early senescence of Pelargonium in vitro by decreasing its sensitivity to ethylene.     

Functional analysis of an indole-diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.     

Screening of the human tumor necrosis factor-alpha (TNF-α) gene promoter polymorphisms by PCR–DGGE analysis

We have designed a new PCR–DGGE technique that enables detection of base changes in the TNF-α gene promoter. Screening of 130 samples from Spanish children has shown that this technique accurately detects the altered band patterns induced by the presence of the polymorphisms at positions −376, −308, −238 and −163 of the promoter sequence. Although further analysis are needed to fully characterise the alterations detected, we believe that this PCR–DGGE technique is a rapid and sensitive first approach to the genetic characterisation of the TNF-α promoter.     

An EcoRI RFLP in the 5′ region of the human NF1 gene

Von Recklinghausen neurofibromatosis or type l neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.