Identification of a nonframeshift 84-bp deletion in exon 13 of the cystic fibrosis gene.

Cystic fibrosis (CF) is the most frequent autosomal recessive inherited disorder in Caucasian populations. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified an 84-bp deletion in exon 13 of the CFTR gene, detected by DNA amplification and direct sequencing of 500 bp of the 5' end of exon 13. The deletion was in the maternal allele of a CF patient bearing the delta F508 deletion in the father's allele. The same 84-bp deletion could also be detected in the patient's mother. The deletion spanned from a four-A cluster in positions 1949-1952 to another four-A cluster in positions 2032-2035, including 84 bp which correspond to codons 607-634 (1949del84). The reported mutation would result in the loss of 28 amino acid residues of the R domain of the CFTR protein.     

Indirect role of adenylate cyclase and cyclic AMP in chemotaxis to phosphotransferase system carbohydrates in Escherichia coli K-12.

Most strains of Escherichia coli K-12 lacking the enzyme adenylate cyclase showed normal chemotaxis toward carbohydrates taken up and phosphorylated by the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system. The normal reaction was observed even in the absence of externally added cyclic adenosine 3',5'-phosphate, provided that the enzyme II chemoreceptors and the flagella were synthesized. In the CA8306 series of strains, however, the cya-854 deletion abolished chemotaxis toward phosphotransferase system carbohydrates even though growth on and transport of these carbohydrates were not affected. This abnormal phenotype was due to the presence of a specific mutation in strain CA8306 which mapped in or close to the crp locus and apparently prevented expression of a hitherto unidentified molecule involved in enzyme II-mediated signal transduction. This molecule is neither a pts protein nor a cyclic adenosine 3',5'-phosphate-binding protein.     

Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Immunoglobulin D switching can occur through homologous recombination in human B cells.

Prototypical class switching in mouse and human immunoglobulin heavy chains occurs through recombination of tandem blocks of short repeats located 5' to each heavy chain constant region (CH) except C delta. Deletion of C mu in immunoglobulin D (IgD)-secreting murine plasmacytomas occurs illegitimately. We demonstrate here that in human IgD-secreting myeloma cells freshly isolated from patient bone marrow and in normal peripheral blood B lymphocytes, an IgD switch can occur through homologous recombination of a direct repeat consisting of a 442-bp sequence 1.5 kbp 3' of the JH complex and a 443-bp sequence that is duplicated almost perfectly (96% similarity) 1.7 kbp 5' of the C delta gene (442/443-base-pair [bp] repeat). This homologous recombination mechanism is not exclusive for IgD switching, since C mu deletion endpoints in two established IgD-secreting myeloma cell lines fall outside the 442/443-bp repeat. The 442/443-bp mediated recombination shows cell type specificity, and we propose that it represents a unique mode for increased levels of IgD secretion in humans.     

RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes.     

Generation of deletions in the 3'-flanking sequences of the Escherichia coli crp gene that induce cyclic AMP suppressor functions

The crp structural gene and its 3'-flanking sequences were subcloned into M13mp8, and in vitro deletions were constructed in both the 5' and 3' ends of the gene by using Bal 31 nuclease. Deletions ranged in size from 24 to 250 base pairs at the 5' end of crp. Sixteen deletions generated at the 3' end of the gene ranged in size from 133 to 675 base pairs. The majority of deletions extended into the crp structural gene. Another class of deletions, i.e., delta crp-4, delta crp-17, and delta crp-2, had endpoints extending in the 3'-flanking sequences external to the crp structural gene. Deletions were subcloned into pBR322 and transformed into the Escherichia coli cya crp deletion strain NCR438. Transformants containing plasmid pBM4 with the delta crp4 mutation, a deletion of 133 base pairs, were cyclic AMP independent. Strain NCR440 harboring this plasmid expressed beta-galactosidase and threonine dehydratase activities and fermented lactose, ribose, arabinose, and xylose in the absence of exogenous cyclic AMP. The delta crp-4 mutation also caused strain NCR440 to be hypersensitive to exogenous cyclic AMP. The cylic AMP receptor protein expressed in maxicells from pBM4 carrying the delta crp-4 mutation comigrated with the wild-type protein on electrophoretic gels. The delta crp-4 mutation demonstrates that sequences distal to the crp structural gene can mediate cyclic AMP suppressor functions.     

Nucleotide sequence of the Belgian G gamma+(A gamma delta beta)0-thalassemia deletion breakpoint suggests a common mechanism for a number of such recombination events

Various types of thalassemia or hereditary persistence of fetal hemoglobin (HPFH) are caused by deletions at the human beta-globin gene cluster. Many of these molecular lesions show a clear clustering as far as size and location of their breakpoints are concerned. This might indicate common recombination mechanisms responsible for the generation of these deletions. The Belgian G gamma+(A gamma delta beta)zero-thalassemia results from a large deletion spanning the beta-globin gene cluster 3' of the A gamma gene. The extent of this deletion, analyzed by field-inversion gel electrophoresis, is approximately 50 kb and is very similar to that of the Indian HPFH (G gamma A gamma HPFH III) previously characterized by P. S. Henthorn et al. (1986). Proc. Natl. Acad. Sci. USA 83: 5194-5198. Isolation of the deletion junction of the Belgian G gamma+(A gamma delta beta)zero-thalassemia by means of inverse polymerase chain reaction confirmed a very close relationship between these two independent deletions. The 3' breakpoint of the Belgian deletion is located at the midpoint of a 160-bp palindrome, only four nucleotides 5' from the correspondent endpoint of the Indian HPFH.     

Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA.     

Physical mapping of the globin gene deletion in (delta beta (0)) -thalassaemia.

We have constructed a physical map of restriction endonuclease cleavage sites in the (delta (+) beta)-globin gene region in the DNA of patients with (delta beta(0))-thalassaemia. This map shows that a 10 kb deletion has occured in (delta beta (0))-thalassaemia to remove the entire beta-globin gene and the 3' portion of the delta-globin gene. The 5' terminus of the deletion is in the large intron of the delta-globin gene and the 3' terminus 1.8 kb to the 3'-side of the beta-globin gene. A similar deletion of about 7 kb has been described previously in the DNA of patients with Hb Lepore; the 5' terminus of the deletion is also in the delta-globin gene but the 3' terminus is in the beta-globin gene. Comparison of the foetal (gamma) globin gene expression in adults with (delta beta(0))-thalassaemia and Hba Lepore suggests that the 3' extragenic regions of the beta-globin gene contain DNA sequences involved in the regulation of gamma-globulin gene expression.     

Functional dissection of the cis-acting sequences of the Arabidopsis transposable element Tag1 reveals dissimilar subterminal sequence and minimal spacing requirements for transposition

The Arabidopsis transposon Tag1 has an unusual subterminal structure containing four sets of dissimilar repeats: one set near the 5' end and three near the 3' end. To determine sequence requirements for efficient and regulated transposition, deletion derivatives of Tag1 were tested in Arabidopsis plants. These tests showed that a 98-bp 5' fragment containing the 22-bp inverted repeat and four copies of the AAACCX (X = C, A, G) 5' subterminal repeat is sufficient for transposition while a 52-bp 5' fragment containing only one copy of the subterminal repeat is not. At the 3' end, a 109-bp fragment containing four copies of the most 3' repeat TGACCC, but not a 55-bp fragment, which has no copies of the subterminal repeats, is sufficient for transposition. The 5' and 3' end fragments are not functionally interchangeable and require an internal spacer DNA of minimal length between 238 and 325 bp to be active. Elements with these minimal requirements show transposition rates and developmental control of excision that are comparable to the autonomous Tag1 element. Last, a DNA-binding activity that interacts with the 3' 109-bp fragment but not the 5' 98-bp fragment of Tag1 was found in nuclear extracts of Arabidopsis plants devoid of Tag1.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).     

Analysis of the cya locus of Escherichia coli

A 9500-bp DNA segment containing the adenylate cyclase gene (cya) of Escherichia coli has been isolated and analyzed. Four large proteins are encoded within this fragment - the adenylate cyclase protein (92 kDal), two proteins of unknown function (37 and 32 kDal), and a part of the uvrD-coded protein. Various truncated adenylate cyclase proteins, made from cya genes having as much as 60% of their carboxy-terminal end deleted, are sufficient to complement cya- hosts. When these truncated cya genes are present on a multicopy plasmid in a cya- host, the synthesis of beta-galactosidase is still regulated by glucose. The "maxicell" technique was used to visualize the four proteins encoded by this region and some of the truncated adenylate cyclase proteins.     

Recombination between two 14-bp homologous sequences as the mechanism for gene deletion in factor IX Seattle 1.

Factor IXSeattle 1 is a 10-kb intragenic deletion identified in a family that has hemophilia B. By sequencing across the site of the deletion, we discovered at the deletion junction a 13-bp sequence (5' . . . TAGAA-GTTCACTT . . . 3') that was homologous to two 14-bp sequences 10 kb apart in introns D and F of the normal factor IX gene. The presence of these homologous sequences in two different regions of the normal gene allows us to propose that genetic recombination has occurred between the sequences, resulting in the gene deletion. The precise recombination site was able to be localized to one of 5 bp (5' . . . AGTTC . . . 3') in the middle of the homologous sequences. The exact length of the deletion is 10,000 bp.     

Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon.

Intramolecular transposition by an engineered derivative of the transposon gamma delta (Tn1000) is described. This 1-kb element contains inverted repeats of the 40 bp of the delta end of gamma delta, bracketing a kan gene, but it contains no resolution site. Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-gamma delta element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA [rpsL]) in a 13.2-kb three-component plasmid (a heterotrimer). Selection for loss of function of a single contraselectable gene yielded inversions and deletions. Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-gamma delta inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated. Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb. The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it. This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.