Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.     

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.     

A highly repeated retropseudogene-like sequence in DNA of the redbreasted merganser (Mergus serrator)

Two highly repeated nucleotide sequences (RBMI and RBMII) cloned from an EcoRI digest of DNA of the redbreasted merganser (Mergus serrator) account for approx. 5 to 10% of the DNA of M. serrator and the closely related Mergus merganser. Complete DNA digestion of seven members of the Mergini with EcoRI produces distinct, relatively species-specific patterns of a few high-Mr (greater than 1.5 kb) fragments of RBMI-like material. In such digests RBMII forms ladder-type patterns with monomers of approx. 200 bp. The sequence of a cloned 2.6-kb RBMI fragment from M. serrator contains several extended (up to 70 bp) and modified poly(dA) sequences, two open reading frames in opposite orientation to the longest poly(dA) sequence and two direct 10-bp repeats suggesting that RBMI is a rearranged retropseudogene-like element.     

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.     

Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage

A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H. Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied. Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage. Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment. When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA. The sum of the fragment lengths equaled the length of the mRNA. Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA. With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.     

Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats.

The plasmid mini-Rts1, consisting of an EcoRI/HindIII fragment of about 1.8 kilobases (kb), contains an incompatibility determinant in its EcoRI/AccI region (0.5 kb). The nucleotide sequence of this incompatibility fragment was determined. A most striking feature of the sequence is the presence of five 24-base pair direct repeats. Four out of the five repeating units, which are contained in a 0.2-kb EcoRI/HincII fragment, were cloned en bloc in pACYC184 and found to express Rts1-specific incompatibility. In addition, the copy number of the mini-Rts1 plasmid appeared to be increased threefold upon removal of the 0.2-kb incompatibility region (incI) from the plasmid. This deletion derivative of mini-Rts1, as well as mini-Rts1, was maintained stably at 37 degrees C, but was cured at a high frequency at 42 degrees C. A possible role of the repeated nucleotide sequence was discussed. By subcloning the mini-Rts1 DNA, a second inc determinant (incII) was found on the AccI fragment, which is contiguous to the 0.5-kb EcoRI/AccI fragment.     

Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.     

The productive gene for alpha-H chain disease protein MAL is highly modified by insertion-deletion processes

alpha-H chain diseases (HCD) is a human lymphoproliferative disorder, characterized by the production of truncated alpha-Ig H chains, without associated L chains. In this study, we have analysed the serum protein, the alpha-HCD mRNA and the rearranged alpha-HCD gene from the leukemic cells of a patient (MAL) with alpha-HCD. The abnormal MAL serum Ig consisted of short alpha 1-chains, lacking VH and CH1 domains (only CH2 and CH3 domains were present). The alpha-HCD mRNA (1.2 kb) was shorter than a normal alpha-mRNA (2 kb); the corresponding cDNA had sequences for the leader, a 84-bp sequence of unknown origin and the CH2 and CH3 exons. The establishment of the sequence of the productive alpha-HCD MAL allele revealed two major deletions; that of the VH region as well as that of the CH1 region. The JH region is altered by multiple mutations, small insertions and a duplication of the psi JH3 region. A large insert (INS1), of 360 bp (containing the 84 bp exon found in the cDNA), replaces the deleted VH region. INS1 is non-Ig related and apparently of nongenomic origin. A large second insert (509 bp), is located between the enhancer and the switch region. Insert2 contains repetitive non-Ig-related sequences and a small Ig-related sequence. All these alterations resulted in an abnormal mRNA, which comprises the leader, a 84-bp alien exon derived from INS1 and the CH2 and CH3 exons of the alpha 1-gene.     

Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket,Acheta domesticus (Orthoptera:Gryllidae)

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.     

Long tandem arrays of complex repeat units in Chironomus telomeres.

A cloned 340-bp DNA fragment excised by EcoRI from the Chironomus pallividittatus genome has been localized to the telomeres by in situ hybridization as well as to connectives between telomeres. No hybridization was observed in other regions of the chromosomes. Another cloned EcoRI fragment, 525 bp long has also been studied. This represents a partial duplication of the 340-bp sequence. Genomic blot hybridization experiments show that the 340-bp sequence is a representative monomeric unit of tandemly repeated arrays which account for 1.2% of the Chironomus genome, on average 300 kb per telomere. The repeat unit contains two types of subrepeats each present twice per repeat unit. Northern blot hybridization experiments show that the telomere-associated sequences are transcribed into a discrete RNA species approximately 20 kb in size. The evolution of this telomere-associated DNA is discussed.     

Physical and genetic analyses of the Inc-I alpha plasmid R64

A 126-kilobase (kb) physical and genetic map of the Inc-I alpha plasmid R64 was constructed by using the restriction enzymes, BamHI, SalI, XhoI, HindIII, and EcoRI. The replication (Rep) and incompatability (Inc) functions of this plasmid were located in a 1.75-kb segment of an EcoRI fragment, E10 (3.3 kb). In addition, the genes determining growth inhibition of phage BF23 (Ibf), suppression of dnaG ( Sog ), resistance to tetracycline (Tetr), and resistance to streptomycin ( Strr ) were located on the 5.5-kb HindIII-XhoI fragment, the 8.1-kb EcoRI fragment (E5), the 4.6-kb HindIII fragment (H8), and the 4.1-kb HindIII fragment (H10), respectively. The map of R64 was compared with that of ColIb, which belongs to the Inc-I alpha group.