There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoesaconformationalchange,causingtemperaturedependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.The constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation in humans. Its recurrent nature implicates a specific genomic structure at the t(11;22) breakpoints. Analyses of numerous independent t(11;22) cases have localized the breakpoints within palindromic AT-rich repeats (PATRRs) 1 on 11q23 and 22q11 (1-4). Most 11;22 translocations show breakpoints at the center of the PATRRs, suggesting that the center of the palindrome is susceptible to double-strand breaks, leading to the translocation (5, 6). Indeed, translocation-specific PCR detects a high frequency of de novo t(11;22)s in normal sperm samples (7).The breakpoints on 22q11 are located within one of the unclonable gaps in the human genome (8, 9). Extensive screening of YAC/BAC/PAC libraries has not been successful in cloning this breakpoint region. However, experimentally derived sequences from numerous t(11;22) junction fragments demonstrate that the 22q11 breakpoints reside within a larger PATRR. The breakpoints of a variety of translocations involving 22q11 cluster within this region, suggesting that the 22q11 PATRR is highly unstable (10 -13). More recently, molecular cloning of translocation breakpoints has demonstrated similar palindromic sequences on partner chromosomes, such as 17q11, 4q35

Hypoglycosylation and reduced laminin-binding activity of α-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated α-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact α-dystroglycan, and solid-phase assays determined laminin binding levels to be ∼50% of normal. In contrast, intact α-dystroglycan is undetectable in the dystrophic Largemyd mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact α-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of α-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of α-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of α-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

SUMMARYThe relative contribution of polymorphonuclear leukocytes and macrophages in the early protection against intranasal infection of mice with influenza virus was investigated. Virus multiplication in the lung in the early phase of infection with less than 1.5 x 10 3 plaque-forming units was enhanced by X-ray irradiation. The intranasal administration of carrageenan did not influence the titre of virus. However, when mice were infected with 1.5 x 10 4 plaque-forming units, the virus titre was elevated by intranasal administration of carrageenan as well as by X-ray irradiation, but not by intraperitoneal administration of carrageenan. The intranasal administration of carrageenan not only inhibited the phagocytic activity of alveolar macrophages but also enhanced susceptibility to the virus. On the other hand, polymorphonuclear leukocytes were capable of phagocytosing the virus in vitro and were non-permissive for virus infection. Neutralizing antibody and interferon were not detectable in the early stage of the infection. These results suggested that polymorphonuclear leukocytes (Xray-sensitive, carrageenan-resistant) were the cells primarily responsible for early protection in influenza virus infection and that after infection with a high dose of the virus alveolar macrophages (X-ray-resistant, carrageenan-sensitive) also played a protective role in the early phase.

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.

Translocation is one of the most frequently occurring human chromosomal aberrations. Balanced carriers usually manifest no phenotype but experience problems with reproduction. These include infertility, recurrent abortion, and offspring with chromosomal imbalance. The constitutional t(11;22)(q23;q11) is a balanced translocation between chromosomes 11 and 22, with breakpoints at bands 11q23 and 22q11. It is the only known recurrent non-Robertsonian translocation and represents a good model for studying translocations in humans (1). The recurrent nature of this translocation prompted us to examine t(11;22) breakpoints for a specific genomic structure. The analysis of many unrelated t(11;22) cases revealed that the breakpoints occur within palindromic AT-rich repeats (PATRRs) on 11q23 and 22q11 (PATRR11 and PATRR22) (2-4). The majority of the breakpoints are localized at the center of the PATRRs, suggesting that the center of the palindrome is susceptible to double-strand breaks (DSBs), thereby inducing illegitimate chromosomal rearrangement (3,5). Recent findings of PATRRlike sequences at the breakpoints of other translocations support the possibility that palindromemediated chromosomal translocation is a general pathway for human genomic rearrangements (6-8).The PATRR11 is variable in size in normal healthy individuals ( Fig. 1 and table S1). The most common allele is a ~450-base pair (bp) PATRR11 (L-PATRR11) that forms a nearly perfect palindrome (5). Several types of short variants were identified (S-PATRR11) that appear to be derived from the longer version primarily by deletion near the symmetric center of the palindromic structure. We can classify the S-PATRR11s into four groups. The most frequent 350-bp variant, S1-PATRR11, has a 50-bp deletion at both of the palindromic arms but still remains completely symmetrical. S2-PATRR11 has an asymmetric deletion at its center, but the new center manifests a symmetric palindrome. S3-PATRR11 does not possess palindromic features by virtue of a deletion at the center of the palindrome. We identified a rare 434-bp S-PATRR11, which sustained an asymmetric central deletion followed by the insertion of an ATrich sequence of unknown origin (S4-PATRR11). We also identified another rare allele with a duplication of the proximal arm, which constitutes a 603-bp asymmetric palindrome (EL-PATRR11). On the basis of the palindrome-mediated mechanism of the translocation, it is reasonable to hypothesize that the polymorphism of the PATRR11 could affect the frequency of de novo t(11;22) translocations.