Cloning the human major histocompatibility complex in YACs.

To clone the human major histocompatibility complex (MHC), 53 YACs, with an average size of 490 kb, were isolated and characterized from the CEPH YAC library. These YACs were organized in a single large contig covering more than 4000 kb. Furthermore, a complete physical map of the previously uncloned HLA class I region was established from partial and/or total digestions of 15 YACs spanning 2000 kb. This resulted in the establishment of the first YAC contig that spans the entire MHC region and constitutes an essential step in the isolation of all of the genes present in the region.

A YAC contig in 6p23 based on sequence tagged sites.

A yeast artificial chromosome (YAC) contig located in 6p23 and spanning roughly 2.5 Mb has been constructed from the content of 10 sequence tagged sites (STSs) for YAC clones in 66 yeast colonies. Nine of the STSs have been genetically mapped in CEPH families. The order of STSs mapped with the contig is consistent with that of the genetic map. The order of loci that did not recombine with each other on the genetic map was inferred from the contig. Various regions of the contig are covered by multiple YAC clones that complement observed STS deletions. The STS for the CAG repeat sequence contained in the gene for spinal cerebellar ataxia 1 (gene symbol SCA1) is localized in the contig. It is likely that this gene is located in 6p23. The frequency of chimeric YAC clones in this contig is 35%. Eleven yeast colonies were found to carry two or more YACs. YAC subclones from some of these colonies showed size variation, and for several subclones, evidence consistent with deletion of a sequence tagged site.

Recombinations in individuals homozygous by descent localize the Friedreich ataxia locus in a cloned 450-kb interval.

The locus for Friedreich ataxia (FRDA), a severe neurodegenerative disease, is tightly linked to markers D9S5 and D9S15, and analysis of rare recombination events has suggested the order cen-FRDA-D9S5-D9S15-qter. We report here the construction of a YAC contig extending 800 kb centromeric to D9S5 and the isolation of five new microsatellite markers from this region. In order to map these markers with respect to the FRDA locus, all within a 1-cM confidence interval, we sought to increase the genetic information of available FRDA families by considering homozygosity by descent and association with founder haplotypes in isolated populations. This approach allowed us to identify one phase-known recombination and one probable historic recombination on haplotypes from Réunion Island patients, both of which place three of the five markers proximal to FRDA. This represents the first identification of close FRDA flanking markers on the centromeric side. The two other markers allowed us to narrow the breakpoint of a previously identified distal recombination that is > 180 kb from D9S5 (26P). Taken together, the results place the FRDA locus in a 450-kb interval, which is small enough for direct search of candidate genes. A detailed rare cutter restriction map and a cosmid contig covering this interval were constructed and should facilitate the search of genes in this region.

Relationship between Charcot-Marie-Tooth 1A and Smith-Magenis regions. snU3 may be a candidate gene for the Smith-Magenis syndrome.

The juxtacentromeric region of the human chromosome 17 short arm (17p11.2-p12) contains genes involved in the Charcot-Marie-Tooth type 1A disease (CMT1A) and the Smith-Magenis syndrome (SMS). CMT1A is associated with a duplication of a short segment whereas SMS is linked to microdeletions, extending toward the centromere. We describe the construction and analysis of a 5 Mb YAC contig spanning the CMT1A duplicated segment and the distal part of four SMS microdeletions. We concluded that the YAC contig contains about 1Mb of genomic DNA which is deleted in the four SMS patients analysed. Moreover two YACs contain both STS deleted in SMS (U3) and STS duplicated in CMT1A (5H5), but the proximal breakpoint associated with the CMT1A duplication is not the same as the distal SMS breakpoint we studied. Finally we located five new STS in SMS deletion. Two of them, a microsatellite (D17S805(23)) and the gene coding for small nuclear RNA U3, have been localized in the contig we described. We may also note that snU3 is the first expressed sequence localized in an SMS deletion so far. The possible participation of this gene in the SMS phenotype is discussed.

Construction of a yeast artificial chromosome contig spanning the pseudoautosomal region and isolation of 25 new sequence-tagged sites.

Thirty-one yeast artificial chromosomes (YACs) from the human pseudoautosomal region were identified by a combination of sequence-tagged site (STS) screenings and colony hybridizations, using a subtelomeric interspersed repetitive element mapping predominantly to the pseudoautosomal region. Twenty-five new pseudoautosomal STSs were generated, of which 4 detected restriction fragment length polymorphisms. A total of 33 STSs were used to assemble the 31 YACs into a single contiguous set of overlapping DNA fragments spanning at least 2.3 megabases of the pseudoautosomal region. In addition, four pseudoautosomal genes including hydroxyindole O-methyltransferase have been positioned on this set of fragments.

Continuum of overlapping clones spanning the entire human chromosome 21q.

A continuous array of overlapping clones covering the entire human chromosome 21q was constructed from human yeast artificial chromosome libraries using sequence-tagged sites as landmarks specifically detected by polymerase chain reaction. The yeast artificial chromosome contiguous unit starts with pericentromeric and ends with subtelomeric loci of 21q. The resulting order of sequence-tagged sites is consistent with other physical and genetic mapping data. This set of overlapping clones will promote our knowledge of the structure of this chromosome and the function of its genes.

Mapping the whole human genome by fingerprinting yeast artificial chromosomes.

Physical mapping of the human genome has until now been envisioned through single chromosome strategies. We demonstrate that by using large insert yeast artificial chromosomes (YACs) a whole genome approach becomes feasible. YACs (22,000) of 810 kb mean size (5 genome equivalents) have been fingerprinted to obtain individual patterns of restriction fragments detected by a LINE-1 (L1) probe. More than 1000 contigs were assembled. Ten randomly chosen contigs were validated by metaphase chromosome fluorescence in situ hybridization, as well as by analyzing the inter-Alu PCR patterns of their constituent YACs. We estimate that 15% to 20% of the human genome, mainly the L1-rich regions, is already covered with contigs larger than 3 Mb.

Isolation of chromosome 21-specific yeast artificial chromosomes from a total human genome library.

A new approach for the isolation of chromosome-specific subsets from a human genomic yeast artificial chromosome (YAC) library is described. It is based on the hybridization with an Alu polymerase chain reaction (PCR) probe. We screened a 1.5 genome equivalent YAC library of megabase insert size with Alu PCR products amplified from hybrid cell lines containing human chromosome 21, and identified a subset of 63 clones representative of this chromosome. The majority of clones were assigned to chromosome 21 by the presence of specific STSs and in situ hybridization. Twenty-nine of 36 STSs that we tested were detected in the subset, and a contig spanning 20 centimorgans in the genetic map and containing 8 STSs in 4 YACs was identified. The proposed approach can greatly speed efforts to construct physical maps of the human genome.

Tumour necrosis factor, IL-1 and IL-6 in bronchoalveolar washings and their in vitro production during Nippostrongylus brasiliensis infection.

Bronchoalveolar washings (BAW) were obtained from rats primarily infected with N. brasiliensis during the early infection stage that coincides with the lung passage of the parasite and the recruitment of inflammatory cells. BAW were tested for IL-1, IL-6 and tumour necrosis factor (TNF) activities. We found that IL-1 production occurred only on day 1 post infection and ceased thereafter. IL-6 activity was present as from day 1 with a maximum on day 3 post infection and then returned to its normal levels on day 5 post infection. TNF activity was not recovered in BAW at any time of the early infection. Results obtained from the in vitro culture of BAW-adherent cells demonstrated that on day 1 post infection IL-1, but also large amounts of TNF were produced spontaneously, whereas IL-6 was continuously released. Bacterial lipopolysaccharide (LPS) stimulation of the cell culture resulted in an amplification of the cytokine production. Our results suggest that pulmonary cytokines detected in BAW were at least in part produced by alveolar macrophages. Furthermore, the kinetics of IL-1, TNF and IL-6 production show that these monokines are induced at different times during the course of infection, suggesting that cytokine production may follow different regulation patterns during the early phase of N. brasiliensis infection.

The CEPH YAC library.

Because of their large size, YACs provide is a powerful tool for physical mapping studies of complex genomes. As it will be advantageous to have genomic libraries of clones with large inserts for analyzing megabase sized regions of the human genome, we have investigated a number of parameters in order to increase the insert size of the YACs. We constructed a genomic library currently containing more than 85,000 YAC clones. Mean sizes of YACs produced at several stages of construction of the library range from 430 kb to 1,200 kb, representing 13 haploid equivalents of the human genome. This library was organized in order to allow rapid screen of YACs for large scale physical mapping of the human genome.

Basophil sensitivity and reactivity to monoclonal anti-human IgE after in vitro sensitization with human myeloma IgE.

Leucocytes from human peripheral blood were acid eluted and sensitized in vitro with increasing concentrations of radiolabelled myeloma IgE. This sensitization step was performed with or without 30% IgE depleted serum. After the IgE binding, cells were washed and submitted to a challenge with monoclonal anti-IgE for the determination of the cellular sensitivity and reactivity in a histamine release assay. A sample of each of the sensitized cells was analyzed for its radioactivity and the number of basophils quantified, thus allowing the determination of the mean number of IgE molecules per basophil. Raising the IgE concentrations in the sensitization procedure led to an increase of the IgE on the basophil membrane, and to a concomitant elevation of the cell sensitivity. The presence of serum during the binding of IgE onto the cells lowers slightly the binding of IgE to the basophils but decreases strongly the cellular reactivity.

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate.

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.

Passive in vitro sensitization of human basophils with Dermatophagoides farinae specific IgE.

Basophils from allergic or non-atopic donors were depleted for their native membrane IgE by acid treatment and then passively sensitized by Dermatophagoides farinae specific IgE containing sera. Histamine release experiments were performed with a highly purified allergen (Df 11) on native cells, acid treated cells and passively sensitized cells. In the sensitization procedure, the quantity of the basophil-bound serum IgE is dependent on the concentration of the sensitizing serum IgE and the histamine release capacity is specifically acquired. It is shown that after passive sensitization basophil membrane IgE density as well as basophil sensitivity to Df 11 in histamine release experiments depend on the ratio [Df 11 specific IgE/total IgE] in the sensitizing serum.