Identification of a hereditary pancreatitis mutation in four West Virginia families.

Hereditary pancreatitis (HP) is the second most common cause of chronic childhood pancreatitis in the United States. Mutations in the cationic trypsinogen gene on chromosome 7 are known to cause HP. We identified four families in West Virginia with symptoms consistent with HP. To determine whether members of these families had defects in the trypsinogen gene, we tested for linkage between the HP gene and simple tandem repeat markers on chromosome 7q and screened for a specific mutation in the cationic trypsinogen gene. Two-point linkage analysis indicated that the disease gene is closely linked to three 7q markers (D7S661, D7S2511, and D7S1805). Restriction fragment length polymorphism analysis showed that all clinically affected members and nonpenetrant carriers from the four families carried a G to A mutation in the third exon of the trypsinogen gene. These findings indicate that this mutation is the cause of HP in the families in our study. The observation that most individuals who carry the mutation have symptoms of HP is consistent with the high but incomplete penetrance of the trait. The presence of a single mutation and a common linked haplotype indicates that the defective allele arose in an ancestor common to all four families.

Expression and function of gamma delta- and alpha beta-T cell receptor heterodimers on human somatic T cell hybrids.

T cell somatic hybrids were obtained by fusion of human tetanus toxoid-specific gamma + delta + T cells and a T cell lymphoma cell line that expresses beta-chain but not alpha-chain transcripts. The hybrids simultaneously and independently expressed alpha beta and gamma delta TCR heterodimers on the cell surface without any significant differences in the level of expression. No heterodimers containing alpha delta-, beta delta-, beta gamma-, and alpha gamma-chains were transported to the cell membrane, indicating a chain specificity in dimer formation. The presence of productively rearranged gamma- and delta-alleles in the hybrid cells and immunoprecipitation of an identical type of TCR-gamma delta from both hybrid and parental gamma + delta + T cells suggests that TCR-gamma delta on the hybrid cells derives from gamma + delta + T cells. Anti-TCR (TCS-delta 1 or WT31) and anti-CD3 antibodies induced a rapid increase in [Ca2+]i in the double-positive hybrids and their variants positive for either the alpha beta or gamma delta complex. Double positive hybrid cells were refractory to stimulation with anti-CD3 antibody after pretreatment with a mixture of anti-TCR-gamma delta and anti-TCR-alpha beta antibodies but not with either antibody alone indicating the functional independence of the two receptors. However, only gamma delta heterodimer receptor was able to respond to tetanus toxoid presented on autologous APC as measured by induction of the p55 chain of IL-2R on stimulated cells.

The chromosome 11 region flanking the t(11;14) breakpoint in human T-ALL is deleted in Wilms' tumor hybrids.

Human chromosomal band 11p13 has been implicated in T cell malignancies carrying t(11;14)(p13;q11) reciprocal translocations and has also been associated with Wilms' tumor and aniridia in a mechanism characterized by overlapping hemizygous constitutional deletions spanning this region. Using probes derived from the T cell receptor delta gene, we have cloned the chromosomal breakpoint in an acute T cell leukemia (T-ALL). Southern blotting analyses of mouse-human somatic cell hybrids from this human T-ALL sample and Chinese hamster-human somatic cell hybrids derived from Wilms' tumor lines have indicated that the 11p13 locus, tcl-2, juxtaposed to the TCR (T cell receptor) delta locus in T cell leukemia, is within the constitutional deletion of two Wilms' tumor-aniridia cases.

Sequence analysis of the MYC oncogene involved in the t(8;14)(q24;q11) chromosome translocation in a human leukemia T-cell line indicates that putative regulatory regions are not altered.

We have cloned the translocation-associated and homologous normal MYC alleles from SKW-3, a leukemia T-cell line with the t(8;14)(q24;q11) translocation, and determined the sequence of the MYC oncogene first exon and flanking 5' putative regulatory regions. S1 nuclease protection experiments utilizing a MYC first exon probe demonstrated transcriptional deregulation of the MYC gene associated with the T-cell receptor alpha locus on the 8q+ chromosome of SKW-3 cells. Nucleotide sequence analysis of the translocation-associated (8q+) MYC allele identified a single base substitution within the upstream flanking region; the homologous nontranslocated allele contained an additional substitution and a two-base deletion. None of the deletions or substitutions localized to putative 5' regulatory regions. The MYC first exon sequence was germ line in both alleles. These results demonstrate that alterations within the putative 5' MYC regulatory regions are not necessarily involved in MYC deregulation in T-cell leukemias, and they show that juxtaposition of the T-cell receptor alpha locus to a germ-line MYC oncogene results in MYC deregulation.

Identification of the Rts 1 DNA fragment responsible for temperature sensitive growth of host cells harboring a drug resistant factor Rts 1.

We have placed a kanamycin resistance SalI fragment (3.65 Kb) from the drug resistance factor Rts1 into pUC19 and pBR322. These chimeric plasmids containing the kanamycin resistance fragment from Rts1 cause temperature sensitive growth in E coli. The orientation of the kanamycin resistance fragment in the vector plasmids does not influence this phenotype.

The t(8; 14) chromosomal translocation occurring in B-cell malignancies results from mistakes in V-D-J joining.

The reciprocal chromosome translocation, t(8;14), involving the heavy chain locus on chromosome 14 and the c-myc oncogene on chromosome 8 is a characteristic of the B-cell malignancies Burkitt's lymphoma and acute lymphoblastic leukaemia (ALL). We have cloned and sequenced the t(8; 14) breakpoints of an African Burkitt's lymphoma cell line, P3HR-1, and a pre-B cell ALL cell line, 380. In each case the region of chromosome 8 involved has recombined with a JH region on chromosome 14. The two sites of breakage on chromosome 8 lie within 70 base pairs (bp) of one another. At each joining site, sequences homologous to the signal sequences thought to be recognized by the V-D-J recombinase were identified, as were N regions. In B-cell chronic lymphocytic leukaemias (B-CLL) carrying the t(11; 14) chromosome translocation and in follicular lymphomas carrying the t(14; 18) translocation, the V-D-J recombinase is implicated in the mechanism of chromosomal translocations. We speculate that the same enzymatic mechanism is responsible for the t(8; 14) translocations in African Burkitt's lymphoma and pre-B cell ALL.

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.