Riccardo Cortese

A general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera.

A strategy to identify disease‐specific epitopes from phage‐displayed random peptide libraries using human sera is described. Peptides on phage (phagotopes) that react with antibodies present in patient sera are purified from > 10(7) different sequences by affinity selection and immunological screening of plaques. Disease‐specific phagotopes can be identified out of this pool through an ‘antigen independent’ procedure which avails itself only of patient and normal human sera. Using this strategy, we have selected antigenic mimics (mimotopes) of two different epitopes from the human hepatitis B virus envelope protein (HBsAg). We could show that a humoral response to these mimotopes is widespread in the immunized population, suggesting that the strategy identifies phagotopes that have a potential role as diagnostic reagents. Immunization of mice with the selected phagotopes elicited a strong specific response against the HBsAg. These results open new inroads into disease‐related epitope discovery and provide the potential for vaccine development without a requirement for the use of, or even information about, the aetiological agent or its antigens.

Purification of a NF1-like DNA-binding protein from rat liver and cloning of the corresponding cDNA.

NF1‐like proteins play a role in transcription of liver‐specific genes. A DNA‐binding protein, recognizing half of the canonical NF1 binding site (TGGCA) present on the human albumin and retinol‐binding protein genes, has been purified from rat liver. Several peptides deriving from a tryptic digest of the purified protein were sequenced and the sequence was used to synthesize specific oligonucleotides. Two overlapping cDNA clones were obtained from a rat‐liver cDNA library; their sequence reveals an open reading frame coding for 505 amino acids, including all the peptides sequenced from the purified protein. The DNA‐binding domain, most likely located within the first 250 amino acids, is highly homologous to the sequence of CTF/NF1 purified from HeLa cells. Northern analysis reveals several mRNA species present in different combinations in various rat tissues.

Two distinct factors interact with the promoter regions of several liver-specific genes.

A segment of the human alpha 1‐antitrypsin (alpha 1AT) 5′‐flanking region comprising nucleotides −137 to −37 from the start of transcription is sufficient to drive liver‐specific transcription from the homologous alpha 1AT promoter and from the heterologous SV40 promoter. In this paper we characterize two proteins, LF‐A1 and LF‐B1, whose ability to bind wild‐type and mutant alpha 1AT promoter segments correlates with the ability of these segments to activate transcription in vivo. DNase I protection and methylation interference analysis reveals that LF‐A1 recognizes sequences present in the regulatory region of the human alpha 1‐antitrypsin, apolipoprotein A1 and haptoglobin‐related genes. These sequences share a common 5′ TGG/A A/C CC 3′ motif. LF‐B1 binds to the palindrome 5′ TGGTTAAT/ATTCACCA 3′ which is present in the human alpha 1‐antitrypsin gene between positions −78 and −62 from the start of transcription. LF‐B1 also recognizes a related sequence present in the human albumin gene between −66 and −50. These results suggest that LF‐A1 and LF‐B1 are common positive trans‐acting factors which are required for the expression of several genes in the hepatocyte.

LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia.

We have cloned and characterized a mouse cDNA coding for LFB3, a DNA binding protein containing an extra‐large homeodomain. The first 315 amino acids of LFB3 are highly homologous to the DNA binding domain of LFB1, a regulatory protein involved in the expression of several liver‐specific genes. LFB3 is a transcriptional activator which binds to DNA as a dimer and forms heterodimers with LFB1 both in vitro and in vivo. However, LFB3 expression seems not to be directly correlated with the liver‐specific phenotype, since it is detected in dedifferentiated hepatoma cell lines which express neither LFB1 nor several liver‐specific genes. LFB3 expression starts before that of LFB1 during mouse and rat development, and is strongly increased upon retinoic acid induced differentiation of F9 embryonic carcinoma cells. LFB3 and LFB1 are expressed in the epithelial component of many organs of endodermal and mesodermal origin, suggesting that they may play a more general role associated with the differentiation of specialized epithelia.

Cell-specific expression of a transfected human α1-antitrypsin gene

We have cloned the human alpha 1-antitrypsin (alpha 1-AT) gene and identified the promoter and the transcription initiation point. The cloned gene, following transfection, is expressed in a cell-specific manner, being transcribed in a human hepatoma cell line (Hep3B) but not in HeLa cells. We show that the 5 flanking region of the alpha 1-AT gene contains DNA sequences sufficient for efficient transcription in Hep3B but not in HeLa cells. This DNA sequence also activates, in a cell-specific manner, heterologous promoters such as that of SV40; however, the effect is only obtained in one orientation, suggesting that this cis-acting cell-specific element does not share all the features generally associated with enhancers. By cotransfection-competition experiments we also show the existence of a limiting trans-acting factor, essential for the expression of the alpha 1-AT gene in Hep3B cells.

Amino-terminal domain of NF1 binds to DNA as a dimer and activates adenovirus DNA replication.

NF1 is a DNA‐binding protein involved in initiation of adenovirus DNA replication as well as in modulating the rate of transcription initiation of genes containing the sequence TGGCA. We show here that recombinant NF1 expressed via vaccinia virus is transported into the nucleus and binds to its cognate sequences with the same specificity as NF1 purified from HeLa cells. Furthermore, the recombinant NF1 forms oligomers in solution and binds as a dimer to palindromic as well as half‐site sequences. NF1 expressed via vaccinia virus stimulates the initiation of adenovirus replication in vitro. The N‐terminal 240 amino acids of the protein are sufficient for full DNA‐binding activity as well as stimulation of adenovirus replication. By analysis of several NF1 mutants translated in vitro, we also define the minimal DNA‐binding domain and localize the region responsible for DNA binding on the N‐terminal and for oligomerization on the C‐terminal side of this domain.

Two different liver-specific factors stimulate in vitro transcription from the human alpha 1-antitrypsin promoter.

The region from −137 to −2 of the human alpha 1‐antitrypsin (alpha 1AT) promoter directs liver‐specific in vitro transcription. Two cis‐acting elements, A and B, have been identified within this segment by site‐directed mutagenesis. Competition with synthetic oligonucleotides corresponding either to the A or to the B sequence inhibits transcription from the wild‐type promoter in vitro. Cis‐linked A and B elements mediate liver‐specific transcription from a truncated HSV‐TK promoter in vitro. Five different proteins, LF‐A1, LF‐A2, LF‐B1, LF‐B2 and LF‐C, bind to the alpha 1AT promoter in liver extracts. LF‐A1 and LF‐B1 are positive transcriptional factors which bind to the A and B elements respectively. Their absence in spleen provides an explanation for the liver specificity of transcription. A protein similar to LF‐B2 is present in spleen. Binding of LF‐B1 and LF‐B2 to the alpha 1AT promoter is mutually exclusive, suggesting that LF‐B2 might be a repressor.

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the ‘haptoglobin related’ gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000‐fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000‐fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.

Common and interchangeable elements in the promoters of genes transcribed by RNA polymerase III

We have shown that the 34 bp internal control region of the somatic 5S RNA gene from Xenopus borealis can be split into two separable components. A maxigene carrying an insertion between nucleotide 71 and nucleotide 74 of the coding region is actively transcribed in the nucleus of X. laevis oocytes, giving rise to a maxitranscript with initiation and termination points identical with those of the wild-type transcript. The first 11 bases of the 5S RNA gene promoter are shown to be structurally and functionally homologous with the first component (box A) of the promoter for tRNA genes. This was shown by constructing hybrid 5S RNA-tRNAPro and tRNAPro-5S RNA genes that were efficiently transcribed in the X. laevis oocytes. Initiation of transcription appears to be a complex phenomenon in which both components of the internal promoter play a role.

Cis- and trans-acting elements responsible for the cell-specific expression of the human alpha 1-antitrypsin gene.

The 5′ flanking region of the human alpha 1‐antitrypsin (alpha 1‐AT) gene contains cis‐acting signals for liver‐specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1‐AT regulatory region. The expression of the bacterial chloramphenicol‐transacetylase (CAT) gene, fused to a set of alpha 1‐AT 5′ flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non‐hepatocyte) human cell lines. A minimal tissue‐specific element has been identified between the nucleotides −137 and −37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (‐125/‐100) and B (‐84/‐70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the ‘minimal element’; the most active of these is located between positions −261 and −210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF‐A and alpha 1TF‐B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.