Multiple nevoid basal cell carcinoma syndrome associated with congenital orbital teratoma, caused by a PTCH1 frameshift mutation.

Gorlin-Goltz syndrome, or nevoid basal cell carcinoma syndrome (NBCCS), is a rare autosomal dominant disorder caused by mutations in the PTCH1 gene and shows a high level of penetrance and variable expressivity. The syndrome is characterized by developmental abnormalities or neoplasms and is diagnosed with 2 major criteria, or with 1 major and 2 minor criteria. Here, we report a new clinical manifestation associated with this syndrome in a boy affected by NBCCS who had congenital orbital teratoma at birth. Later, at the age of 15 years, he presented with 4 major and 4 minor criteria of NBCCS, including multiple basal cell carcinoma and 2 odontogenic keratocysts of the jaw, both confirmed by histology, more than 5 palmar pits, calcification of the cerebral falx, extensive meningeal calcifications, macrocephaly, hypertelorism, frontal bosses, and kyphoscoliosis. PTCH1 mutation analysis revealed the heterozygous germline mutation c.290dupA. This mutation generated a frameshift within exon 2 and an early premature stop codon (p.Asn97LysfsX43), predicting a truncated protein with complete loss of function. Identification of this mutation is useful for genetic counseling. Although the clinical symptoms are well-known, our case contributes to the understanding of phenotypic variability in NBCCS, highlighting that PTCH1 mutations cannot be used for predicting disease burden and reinforces the need of a multidisciplinary team in the diagnosis, treatment, and follow-up of NBCCS patients.

The coat protein of tobamovirus acts as elicitor of both L2 and L4 gene-mediated resistance in Capsicum.

In Capsicum, the resistance conferred by the L(2) gene is effective against all of the pepper-infecting tobamoviruses except Pepper mild mottle virus (PMMoV), whereas that conferred by the L(4) gene is effective against them all. These resistances are expressed by a hypersensitive response, manifested through the formation of necrotic local lesions (NLLs) at the primary site of infection. The Capsicum L(2) gene confers resistance to Paprika mild mottle virus (PaMMV), while the L(4) gene is effective against both PaMMV and PMMoV. The PaMMV and PMMoV coat proteins (CPs) were expressed in Capsicum frutescens (L(2)L(2)) and Capsicum chacoense (L(4)L(4)) plants using the heterologous Potato virus X (PVX)-based expression system. In C. frutescens (L(2)L(2)) plants, the chimeric PVX virus containing the PaMMV CP was localized in the inoculated leaves and produced NLLs, whereas the chimeric PVX containing the PMMoV CP infected the plants systemically. Thus, the data indicated that the PaMMV CP is the only tobamovirus factor required for the induction of the host response mediated by the Capsicum L(2) resistance gene. In C. chacoense (L(4)L(4)) plants, both chimeric viruses were localized to the inoculated leaves and produced NLLs, indicating that either PaMMV or PMMoV CPs are required to elicit the L(4) gene-mediated host response. In addition, transient expression of PaMMV CP into C. frutescens (L(2)L(2)) leaves and PMMoV CP into C. chacoense (L(4)L(4)) leaves by biolistic co-bombardment with a beta-glucuronidase reporter gene led to the induction of cell death and the expression of host defence genes in both hosts. Thus, the tobamovirus CP is the elicitor of the Capsicum L(2) and L(4) gene-mediated hypersensitive response.

Biological and molecular characterization of P101 isolate, a tobamoviral pepper strain from Bulgaria.

A tobamovirus isolated from pepper crops in Bulgaria has been characterized, and is referred to below as P101. It was closely related to Paprika mild mottle virus (PaMMV) (Dutch isolate), based upon the serological relationship of its coat protein, and the nucleotide sequence analysis of the gene encoding the coat protein and the 3' non-coding region of the viral RNA. The coat proteins of the two isolates differ by two amino acids, and these substitutions may be responsible for the different reactivity of the isolates towards a polyclonal antiserum raised against the virion of the Dutch isolate. The biological behaviour of both isolates was similar in the hosts tested, except in pepper plants where P101 induced delayed and milder symptoms compared with PaMMV, although their accumulation levels were similar. In addition, we investigated the infection pattern of the two isolates in tomato plants. Both isolates accumulated in protoplasts as well as in inoculated leaves, although systemic invasion was limited. This limited spread was not due to activation of defense mechanism(s) in the plant, since the upper uninoculated leaves from P101-infected tomato plants were fully susceptible to challenge inoculation with the virus. Instead, it appears due to a restriction of long-distance movement, that could be overcome in tomato plants co-infected with Tobacco mosaic virus (TMV), but not with either Cucumber mosaic virus or Pepino mosaic virus. The ability of P101 to move systemically in the presence of TMV was not linked to enhanced accumulation of P101 at the cellular level. Thus, a tobamovirus but not the viruses tested from other genera could complement, in trans, the function(s) required for PaMMV to invade the upper uninoculated leaves. Paprika mild mottle virus strain B is proposed as the name for this new isolate.

Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo.

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

Krox-20: a candidate gene for the regulation of pattern formation in the hindbrain.

The mouse gene Krox-20 was isolated on the basis of cross-hybridization with Krüppel, a Drosophila segmentation gene. During recent years, an accumulation of structural, biochemical and expression data has indicated that Krox-20 encodes a transcription factor which may play a key role within a regulatory network involved in pattern formation in the developing hindbrain. The DNA-binding domain of Krox-20 consists of 3 zinc fingers and the protein recognizes a specific GC-rich nucleotide sequence. It can activate the transcription of genes located in the vicinity of this sequence. Krox-20 transcription itself is modulated by growth factors. During formation of the vertebrate central nervous system (CNS), the hindbrain is organized into segmental units, called rhombomeres. Prior to the appearance of morphological segmentation, Krox-20 is expressed in 2 stripes within the hindbrain. Later on, the Krox-20 expression domains match with 2 alternate rhombomeres. These data suggest that Krox-20 may regulate aspects of the segmentation process. The observation of segment-specific expression boundaries for homeobox containing genes in the hindbrain suggests that these genes may also be part of the regulatory network governing pattern formation in this region of the CNS. The possible interactions between Krox-20 and homeobox containing genes as well as the search for other members of the network will be discussed.

The two core sequences of the adenovirus E1A inducible E4 promoter are required for the formation of a specific DNA-protein complex.

We have previously demonstrated that the Adenovirus 2 (Ad2) E4 promoter is activated by an E1A gene product through an inducible enhancer. We now show that several DNA-protein complexes can be identified by gel-shift assay; the formation of one of these complexes involves the two core sequences previously found critical to the promoter activity.

c-myc products trans-activate the adenovirus E4 promoter in EC stem cells by using the same target sequence as E1A products.

Using a short-term transfection assay, we show that the E4 early adenovirus promoter is expressed to a certain extent in undifferentiated F9 and PCC4 cells, which are known to possess cellular E1A-like activity. We have also observed that c-myc products trans-activate the E4 promoter in EC stem cells and HeLa cells. Using 5' deletion mutants of the E4 promoter, we show that the same target sequence is used by c-myc and E1A. This sequence is located between positions -179 and -158 upstream of the cap site and is known to contain an activating transcription factor (ATF)-binding site. Moreover, the basal of level of activity of the deletion mutants. is related to the number of ATF binding sites. We therefore suggest that c-myc is a functional cellular homolog of the viral E1A gene and that it might correspond to one of the cellular E1A-like activities previously described for EC stem cells. We have also observed that only a c-myc plasmid coding for both p67 and 64 proteins, in contrast to one coding for p64 only, is able to trans-activate the E4 and E2A adenovirus promoter, suggesting that the p67 protein plays an essential part in activation.

Transcriptional activation by the E1A regions of adenovirus types 40 and 41.

In order to establish whether the poor growth of the two fastidious adenoviruses types 40 and 41 (Ad40 and Ad41) in HeLa cells is due to a reduced trans-activation by the early region 1A (E1A), we have determined the trans-activating effect of this region on the expression of the chloramphenicol acetyltransferase (CAT) gene controlled by the Ad2 E4 promoter. Cotransfection of HeLa cells with plasmids containing the E1A regions of Ad5, Ad40, and Ad41, respectively, and the CAT gene controlled by the Ad2 E4 promoter showed that activation of the E4 promoter by the E1A regions of Ad40 and Ad41 depends on the same sequence elements of the E4 promoter as activation by the Ad5 E1A gene products. The level of activation, however, is significantly lower. This might partly explain the reduced growth in HeLa cells of the two viruses.

The E4 promoter of adenovirus type 2 contains an E1A dependent cis-acting element.

To study how the E1A polypeptides of adenovirus type 2 regulate transcription, we have constructed chimeric plasmids containing the bacterial gene encoding chloramphenicol acetyl transferase (CAT) under the control of either the wild type or the deleted E4 promoter of adenovirus type 2. Our previous results showed that promoter sequences located upstream from position -158, as measured from the cap site, are essential to the transactivation process. From a new set of deletion mutants, we now show that two regions, located between positions -239 and -218 and between positions -179 and -158, are involved in the E1A transactivation process. The deletion of only one of them does not significantly alter the E1A induction process compared with the wild type. Moreover, we show that these two regions lie within a DNA fragment which possesses the properties of an E1A-inducible "enhancer-like" element. In addition, the DNA fragment which contains this enhancer element is also able to confer the E1A inducibility to a heterologous promoter.

The E4 transcriptional unit of Ad2: far upstream sequences are required for its transactivation by E1A.

We have investigated the effect of the E1A polypeptides of adenovirus 2 on the transcription of the viral E4 region. For this purpose, we have fused the promoter region of the E4 gene to the bacterial gene coding for chloramphenicol acetyl transferase. We have found that transcription from the E4 promoter is increased at least 20 fold in the presence of the E1A region. We have also found that the largest E1A polypeptide is the regulating factor, whereas the shortest has no apparent effect. Deletion of sequences upstream from position -158, as measured from the cap site, reduces the efficiency of the transcription in the presence of E1A by more than 15 fold. An important regulatory domain lies between positions -158 and -179. This domain contains the sequence 5' GGGAAGTGAC 3' which is homologous to the E1A enhancer core sequence. Another similar sequence is also present at position -149.

mRNAs from human adenovirus 2 early region 4.

The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides.

Polyadenylic acid addition sites in the adenovirus type 2 major late transcription unit.

The cytoplasmic mRNAs which are transcribed from the major late adenovirus promoter can be arranged into five 3'-coterminal families, L1 to L5. We have defined the polyadenylation sites of the mRNAs that belong to the five families at the nucleotide level. From the results, the following conclusions can be made. (i) The hexanucleotide sequence AAUAAA is present at the 3' end of all late adenovirus type 2 mRNAs and precedes the site of polyadenylation by 12 to 30 nucleotides. (ii) Between one and three A residues are present in the genomic sequence at the polyadenylation site. (iii) A sequence with the composition (T)n (A)p (T)q (n, p, q greater than or equal to 1) is found 4 to 24 nucleotides beyond all the adenovirus-specific polyadenylation sites except the 3'-coterminal family L4. This sequence is also found beyond many cellular polyadenylation sites. (iv) The L1 and L2 polyadenylation sites are very similar in structure. The other polyadenylation sites show no apparent sequence relationship, except for the hexanucleotide sequence.