Recognition of oligonucleotide-encoded T cell epitopes introduced into a gene unrelated to the original antigen.

We have previously demonstrated that H-2Kd-restricted CTL specific for HLA-CW3 or HLA-A24 can recognize synthetic peptides corresponding to residues 170-182 of the HLA molecules. Synthetic oligonucleotides encoding region 170-182 of CW3 or A24 were inserted into the influenza nucleoprotein (NP) gene. We demonstrate herein that P815 (H-2d) cells transfected with the NP-oligo recombinant genes are specifically lysed by HLA-specific Kd-restricted CTL clones. Our results imply that there must be a high degree of flexibility for the expression of T cell epitopes in different molecular contexts.

Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11.

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.

The FGF-related oncogenes hst and int.2, and the bcl.1 locus are contained within one megabase in band q13 of chromosome 11, while the fgf.5 oncogene maps to 4q21.

Prompted by the observed co-amplification at the DNA level of the int.2 and hst fibroblast growth factor-related oncogenes in some tumor cells, we have investigated the precise localization of these two loci known to reside in band q13 of chromosome 11. We demonstrate by pulsed field gel analysis that these two genes are separated by only 40 kb, locate three HTF islands in their neighbourhood, and show that the bcl.1 locus (translocation breakpoint in B-cell proliferative malignancies) is not more than 1050 kb away. We also show that the fgf.5 gene which belongs to the same family is not part of this cluster and is located in band q21 of chromosome 4.

alpha-Hydroxylation of newly synthesised fatty acids by a soluble fraction from germinating pea.

A soluble fraction from germinating pea (Pisum sativum) seeds alpha-hydroxylated newly-synthesised fatty acids to form alpha-hydroxypalmitic and alpha-hydroxystearic acids. In contrast to fatty acid synthesis from [14C] malonyl CoA, alpha-hydroxylation was inhibited by exogenous phospholipids. alpha-Hydroxylation was optimal at pH 8, required reduced pyridine nucleotides and was inhibited by EDTA and imidazole.

Transformation of LMTK- cells with purified HLA class I genes--IV. A determinant on beta 2-microglobulin is controlled by HLA heavy chain in a mouse-human hybrid complex: a biochemical analysis.

Transformation of LMTK- murine fibroblast cells with purified HLA class I heavy chain genes resulted in the expression of serologically detectable HLA-A3 molecules. Surprisingly, such cells also react with a murine monoclonal antibody specific for a serological determinant notably not expressed by murine but by human beta 2-microglobulin. The human HLA molecules expressed by the transformed cells were characterized on two-dimensional gels. The heavy chain was shown to be associated with a murine beta 2-microglobulin molecule, which could be distinguished from human beta 2-microglobulin by its higher isoelectric point. This heterodimer molecule was immunoprecipitated with the mouse anti-human beta 2-microglobulin monoclonal antibody showing that indeed the complex of mouse beta 2-microglobulin and human heavy chain expresses a human beta 2-microglobulin determinant.

Rat immunoglobulin delta heavy chain gene: nucleotide sequence derived from cloned cDNA.

Rat immunoglobulin delta heavy-chain mRNA has been isolated. RNA blot analysis revealed that this mRNA with a length of 1.8 kb encodes for the secreted form of IgD. The corresponding cDNA was cloned in plasmid pBR322 and its sequence was determined. The hybrid plasmid contains a 775-bp insert comprising a partial C delta 1 sequence and complete C delta H, C delta 3, C delta DC and 3' untranslated sequences. Rat and mouse IgD amino acid sequences show striking homology in C delta 3 and C delta DC regions.

HLA cosmid clones show complete, widely spaced human class I genes with occasional clusters.

To understand the organization of the human leukocyte antigen (HLA) gene region and its relationship to the transplantation antigens expressed at the cell surface we have isolated clones containing HLA class I genes from a cosmid library (Grosveld et al., Gene 13, 227, 1981) constructed with the DNA from an individual of defined haplotype. Most of the cosmids contain a single HLA gene in 30-40 kb of human DNA, indicating that human class I genes are rather widely spaced; two contain two genes and one contains three. Most of these genes appear to be complete; the double or multiple genes are found in the same orientation. Differences in restriction maps are evident but some common features are observed in particular in the 5' half of these genes.

Reduction in cab and psb A RNA transcripts in response to supplementary ultraviolet-B radiation.

The cab and psb A RNA transcript levels have been determined in Pisum sativum leaves exposed to supplementary ultraviolet-B radiation. The nuclear-encoded cab transcripts are reduced to low levels after only 4 h of UV-B treatment and are undetectable after 3 days exposure. In contrast, the chloroplast-encoded psb A transcript levels, although reduced, are present for at least 3 days. After short periods of UV-B exposure (4 h or 8 h), followed by recovery under control conditions, cab RNA transcript levels had not recovered after 1 day, but were re-established to ca. 60% of control levels after 2 more days. Increased irradiance during exposure to UV-B reduced the effect upon cab transcripts, although the decrease was still substantial. These results indicate rapid changes in the cellular regulation of gene expression in response to supplementary UV-B and suggest increased UV-B radiation may have profound consequences for future productivity of sensitive crop species.

Molecular biology of class II genes in man: an introduction.

This introduction attempts to set the stage for the presentation of results on HLA-DR gene cloning and to briefly indicate the basic approaches used and their associated difficulties.

Transformation of LMTK- cells with purified HLA class I gene. VI. Serological characterization of HLA-B7 and AW24 molecules.

Serological characterization of HLA-B7 and HLA-AW24 class I molecules following transfection of murine LMTK- cells with purified HLA class I genes was performed using human alloantisera. Induction by murine alpha interferon of the expression of class I molecules was required to obtain unambiguous identification of these molecules which appear serologically identical to the HLA-B7 and HLA-AW24 molecules expressed at the surface of human peripheral blood lymphocytes of 20 unrelated individuals. Analysis of the transformed cells with 8 different anti-HLA class I monoclonal antibodies results in the definition of 3 separate clusters of antigenic determinants shared by all HLA class I molecules. These studies further suggest the existence of locus-specific serological reactivities associated either with the HLA-A or with the HLA-B and C gene products.

Cytogenetic and physical mapping in the region of the X chromosome surrounding the fragile site.

Seven DNA probes have been mapped within the Xq27-Xq28 region using in situ hybridization, in some cases on chromosomes expressing the fragile site to enhance the resolution. To complement these studies and investigate the relationship between genetic, cytogenetic and physical distance some of these probes were used for large scale mapping using pulsed field gels. Physical linkage was demonstrated between two loci, F9 and MCF2, which are separated by less than 270 kb, and a restriction map extending over 1,300 kb has been generated.

Large-scale expression measurement by hybridization methods: from high-density membranes to "DNA chips".

The vast amount of sequence information becoming available on genes from man and from other species calls for corresponding increases in the rate of collection for data of a more functional nature. Expression measurements often constitute a first step in this direction, and can be performed on a reasonably large scale using highly parallel hybridization methods. Large sets of targets (clones, inserts, oligonucleotides) are hybridized with labeled complex probes prepared from total cell or organ mRNA; under the proper conditions, signals measure the relative abundance of each sequence species, and can be acquired quantitatively. These techniques are presently available in three formats: high-density membranes to be hybridized with radioactive complex probes, microarrays of DNA spots (a miniaturized version of the former technique) using fluorescent complex probes, and oligonucleotide chips that, although developed originally for mutation detection, can be adapted to perform expression measurements. The miniaturized formats clearly represent the future, since they allow higher sensitivity, assay of large numbers of entities and hopefully provide the opportunity to use small amounts of starting material.

Genome programmes: an opportunity for biology.

The development of human molecular genetics in the eighties led to the implementation of projects aimed at a systematic study of the human genetic make-up--the so-called "Genome Projects". These have so far been mainly concerned with genome mapping, both by family studies using polymorphic markers (genetic mapping) and by direct analysis of DNA with suitable fractionation and cloning methods (physical mapping). Major progress has been made in both fields recently. Sequencing per se has remained limited because of technical constraints, although systematic cDNA sequencing has caught on in a large way. National policies are quite diverse, with the USA, the UK, France and Japan being the major players; funding sources range from governments to foundations. Many of the results, methods and concepts obtained or implemented during genome studies can be of great use to "conventional" laboratories, and efficient interfacing between these two worlds is an increasingly important issue.

Differential mRNA expression in untreated and TNF-alpha elicited murine dendritic cells precursors.

We have compared the pattern of gene expression in long term cultured precursor dendritic cells (DC), either untreated (immature) or cultured for two days in the presence of recombinant murine (rm)-TNF alpha (mature). The hybridization signature of complex cDNA probes prepared from total RNA extracted from immature and mature DC were analyzed using a mouse thymic cDNA library, gridded on high density filters. For each clone spotted on the filters, we have measured using an imaging plate device the hybridization signals of the complex probe obtained from immature or mature DC. Comparative analysis of these values allowed us to identify differentially expressed gene products. Our goal is to identify a new set of genes induced or repressed during DC maturation elicited by rmTNF alpha treatment.