1. Volume 1, Issue 6, Pages 773-781 (May 1998)
FRA10B Structure Reveals Common Elements in Repeat Expansion and Chromosomal Fragile Site Genesis 
Duncan R Hewett, Oliva Handt, Lynne Hobson, Marie Mangelsdorf, Helen J Eyre, Elizabeth Baker, Grant R Sutherland, Simone Schuffenhauer, Jen-i Mao, Robert I Richards 
Molecular Cell 
Volume 1, Issue 6, Pages (May 1998)
DOI: /S (00)

2. Figure 1 Positional Cloning of the FRA10B Region
(A) Genetic map of polymorphic markers in the FRA10B region.
(B) YAC contig including the markers D10S597 and D10S88. The location of other markers used to map the extent of each YAC is indicated.
(C) Restriction map of the distal ends of YACs 944H7 and 941C4.
(D) Tile path of lambda subclones from YAC 944H7 and their location (by FISH) with respect to the FRA10B fragile site. Open bars indicate FISH signal proximal to the fragile site; shaded bars indicate distal signal. Subclones with open and shaded bars gave at least one signal on both sides of the fragile site.
(E) Restriction map of lambda subclones (numbered) in the immediate vicinity of FRA10B and the position of these subclones and their restriction fragments with respect to the fragile site as determined by FISH. Open bars indicate FISH signal proximal to the fragile site; shaded bars indicate distal signal.
(F) Restriction fragments used on Southern blots of FRA10B carriers indicating their ability to detect the instability associated with fragile site chromosomes. Open bars indicate constant-sized restriction fragments; hatched bars indicate variable, larger bands in FRA10B individuals.
(G) Structure of the 1.9 kb PstI restriction fragment showing instability in FRA10B chromosomes. Arrows indicate the location of various AT-rich repeats of 16–52 bases in length that were amplified using primers F1 or F2 and R.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2 Southern Blot Detection of Expansion at FRA10B
(A) Expansion associated with FRA10B chromosomes. Southern blot of SacI-digested DNA from FRA10B individuals probed with the 4 kb EcoRI fragment from subclone λ1002. Circles indicate the position of restriction fragments carrying fragile-site-associated amplification. Bracket indicates normal-sized hybridizing restriction fragments. Size markers are indicated in kilobase pairs.
(B) Expansion in a FRA10B homozygote. Southern blot of PstI-digested DNA from two FRA10B heterozygous parents (half-shaded symbol) and their FRA10B homozygous offspring (fully-shaded symbol) probed with the 1.9 kb PstI fragment from subclone λ1002.
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3
Length Polymorphism of the FRA10B AT-Rich Region Revealed by PCR
PCR amplification across the FRA10B-associated unstable region revealed length variation among normal and fragile site alleles. At least fifteen non–fragile site alleles—distinguishable by length alone—were detected and are indicated by numbered arrows. Allele #16 is the smallest allele that gives rise to cytogenetic expression of FRA10B. DNA size markers are indicated in kilobases. Alleles have been grouped into short normal (SN), intermediate (INT), long normal (LN), and FRA10B (EXP) on the basis of size and ability to express the FRA10B fragile site. Allele frequencies for each group are indicated as percentages and were determined by analysis of 170 chromosomes. Artifactual bands (e.g., between allele 12 and 13) were occasionally found in some PCR products and were not reproducible.
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4 Various Repeat Motifs in the FRA10B Unstable Region
Sequence analysis of normal and FRA10B alleles revealed a variety of repeats that could be divided into distinct groups by virtue of their relatedness to one another. The TspI derivate repeats (T), proximal derivate repeats (P), expanded derivate repeats ([e] and [E]), SnaBI derivate repeats (S), and distal derivate repeats (D) are aligned to maximize identity within and between groups. (e) and (E) are related but distinct repeats—all expanded repeats belonging to the (E) group. Sequences that give rise to common restriction enzyme sites (TspI, SnaBI, and NdeI) are boxed.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5
Repeat Motif Composition of Various FRA10B Alleles and Sequence Comparison of FRA10B and FRA16B Expanded Repeat Motifs
(A) Sequence analysis of various “normal” FRA10B locus alleles. Partial sequences were determined for eleven of the alleles in Figure 3 (#1–#4, #6–#8, #11, #12, #14, and #15), the shortest FRA10B allele (#17), and two additional FRA10B individuals (#18 and #19). Sequences were aligned to maximize identity within and between groups. The LN alleles (#14 and #15) and the FRA10B alleles (#17–#19) are from unrelated individuals. The regions of proximal and distal flanking sequence identity limited to FRA10B and LN alleles are boxed. FRA10B alleles are shaded. Question marks indicate undetermined sequence, and dashes represent gaps introduced to maximize alignment. Multiple copies of certain repeats are indicated by brackets, e.g., (T1)3 = T1T1T1. NR indicates nonrepetitive sequence. Relative position and orientation of PCR primers F1, F2, and R are identified by arrows.
(B) Comparison of the FRA16B and FRA10B expanded repeat motifs. A consensus sequence was derived from E repeats found in multiple copies at the extremities of sequencing into the LN and FRA10B alleles. The FRA16B 33mer and the FRA10B 42mer are aligned to demonstrate 26 bases of homology including a 9 bp repeat (black arrows common to all motifs and grey arrows found in some motifs). Hairpin structure can be formed based on the inverted copies of the 9 bp repeat. An identical 12 base sequence at the start of each repeat motif is shaded. (w) indicates that the base can be either A or T.
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 6
Intergenerational and Somatic Instability of the Expanded FRA10B Allele
PCR across the smallest of the FRA10B fragile site alleles in families, showing increase in size upon intergenerational transmission and a doublet-expanded band in one individual indicative of somatic instability ([A], lane 2). Inset boxes show the relevant section of the agarose gel after prolonged electrophoresis. PCR products were stained with ethidium bromide. Size markers are given in kilobase pairs. In (B), black arrows indicate FRA10B alleles, open arrows LN14 alleles. In the pedigrees, half shading indicates the presence of a single FRA10B chromosome. Size ranges for small normal (SN), intermediate (INT), long normal (LN), and expanded (EXP) alleles are indicated.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7 FRA10B Repeat Motif Length and Composition
(A) Ladder PCR of two unrelated FRA10B individuals. A section of an autoradiograph is shown to indicate differences in ladder spacing—7 bands of a longer repeat in one individual span the same distance as 8 repeats in another.
(B) Digestion of FRA10B PCR products with NdeI. Lanes A and D are unrelated individuals; lanes B and C are related, as shown in Figure 6A, lanes 2 and 3. (−) and (+) indicate without and with NdeI digestion, respectively. Arrows indicate NdeI-sensitive FRA10B alleles before (black) and after (white) digestion.
(C) A likely model to explain the observed differences in E repeat composition of the FRA10B allele transmitted between individuals (B) and (C). Shaded arrows represent repeats containing the NdeI recognition site; open arrows are deficient in this site. Gaps indicate an unknown number of additional repeats common to each allele. The exact number of copies by which each allele differs is unknown. Somatic instability in individual (B) is due to differences in NdeI-resistant repeat copy number. The FRA10B allele in individual (C) has fewer NdeI-resistant repeats and a greater number of NdeI-sensitive repeats than either of the FRA10B alleles seen in individual (B).
Molecular Cell 1998 1, DOI: ( /S (00) )