Fig 1.
Illustration of SHS: Sequences of two different genes (coded by red and black bars, respectively) join together.
At, and only at, the junction site (green bar), the two sequences are reversely complementary to each other, say 5’ATCGGT3’ vs 3’TAGCCA5’, while the rest parts of the two genes are not related to each other. In most cases this homologous region is short in length, and thus is coined as short homologous sequence, or SHS.
Table 1.
Primers used in this study.
Fig 2.
Illustration of the six 16S rRNA chimeric RNAs we cloned.
A: The human 16S rRNA ends with 5’GGGTTT3’, followed by the mt-tRNA-leu that starts with 5’GTTAAG3’. The region around the joining site of the two partner sequences in the six 16S rRNA chimeras we cloned is presented, with the 5’ and 3’ partner sequences shown in black and red colors, respectively. The primers used for the cloning are in parenthesis. The two partners overlap at an SHS that is shaded by yellow color. In the top four chimeras, the 5’ and 3’ partner sequences overlap at 5’GGGTTTGTTA3’, which is the 2761-2770th/3221-3230th bps of the mtDNA. In the 5th chimera, the two partner sequences overlap at 5’TCCCT3’ (the 3115-3119th/2617-2621st), whereas in the 6th chimera the two partner sequences overlap at 5’GGAACAAGT3’ (the 2914-2292nd/2602-2610th). All the nt numbers are referred to AY195786.2. All the overlaps are in a mutually reverse-complementary manner. B: PCR was performed with RT product as the template and with the primers indicated. The PCR product was loaded into an agarose gel. M indicatesmarkers of DNA molecular weight, which in B-1 were the 100-bp, 200-bp, 300-bp, 400-bp, 500-bp and 650-bp from the bottom. R in B-1 and B-4 indicates a non-RT control, i.e. a PCR with a diluated RNA sample directly used, without RT, as the template. The numbers 1 to 6 indicate the chimeras shown in A. The chimeric DNA band in the red box was excised out from the gel for purification and cloning. C: Illustration of our hypothesis of how the first four chimeras may be formed as artifacts, because the SHS mis-primes RT or PCR and uses the 1669-2760th nts of the 16S rRNA (or the second strand cDNA) as the template to synthesize a reversely complementary (RC) sequence, resulting in a palindromic or hairpin-like cDNA. Positions of some primers (mtF2006, mtF2581 and mtF2847) on the cDNA are indicated. D: Because of the palindromic nature, a single primer coined as A or B may serve as both forward and reverse primers in PCR to amplify the chimera. Using two primers with the same orientation, say A and B, may produce 3 bands of different sizes primed respectively by the A-A pair, A-B pair, and B-B pair, although sometimes only 2 bands may be discerned easily.
Fig 3.
Cloning an ATP8-sequence containing chimera.
Our 898652R715 primer (the first 20 nts in boldface) has its last 7 nts (underlined) annealed to and mis-priming the amplification of the 569382-569448th-bp region of chr 1 or the 8829-8895th-bp region of the mtDNA (AY195786.2) with 1-nt mismatch (the lowercase letter “g” in boldface) as the 5’ partner. The 3’ partner is the 8360-8521st-bp region of the mtDNA (AY195786.2), with our forward primer at the 3’ terminus (898652F4; the underlined 20 nts in boldface) in a reverse-complementary manner. The 3’ partner may also be the 633501-633694th-bp region of chr 1 with mismatches at 4 nts (lowercase letters in boldface). The two partners overlap at 5’AAATGCCC3’ (underlined, boldfaced, and shaded in grey), which are the 8888-8895th-bps/8360-8367th-bps of the mtDNA in AY195786.2.
Fig 4.
Part of the sequence around the joining site of the six ESTs with mt sequences as both partners.
In each EST, the SHS is shaded in grey color. In the BE898652, the two partner sequences are at the same orientation and overlap at only 2 nts. In the AW516697, AW516528 and EU863790, the two partners overlap at 5’GGGTTTGTTA3’ or its reversely complementary sequence.
Fig 5.
Two different ending sites (shaded, boldfaced and underlined) of 16S rRNA with the lowercase ‘a” belonging to either the RNA or the poly-A tail appended during polyadenylation.
The boldfaced sequence at the 5’ terminus is the mtR2694 (top panel) or the mtF2581 (bottom panel) primer. The boldfaced sequence after the poly-A tail is the newC primer or part of the newA primer.
Fig 6.
Cloning an earlier ending of 16S rRNA with 11 mismatches and a poly-A tail.
A: Using our newA and mtF2006 primers in PCR to amplify the newA-primed RT product from HEK293 cells, we obtained a cDNA that contains the 2006-2274th-bp region of the mtDNA in AY195786.2, followed by a 16-nt sequence (shaded) that has four mismatches (in lowercase letters) to all mtDNA references we can find, and then a poly-A tail (underlined). Our newA primer contains 17 As (shaded), which constitute part of the poly-A tail, and a 23-nt sequence that is our newC primer (boldfaced). The 2006-2274th-bp region contains 11 single-nt mismatches (shaded). B: Alignment of our sequence with BC000845.1, which is a normal mt sequence and is 24-nt longer (shaded) than our sequence before a poly-A tail. Note that within the 2006-2274th-bp region, the 11 mismatches (shaded) occur only at A and C, compared with the BC000845.1 or with all mtDNA references we can find. C: Image of mtDNA isolated from HEK293 cells using the simple method we developed. The mtDNA that migrated much slower than the 12-kb band in the linear DNA markers (M) in a 1% agarose gel was excised from the gel and purified for PCR, cloning and sequencing.
Fig 7.
Cloning of a mouse Bcl2-Nek9 chimeric cDNA.
A: Using Bcl2-L1211 and Bcl2-R2i primers in PCR to amplify an RT product from Ela-mycPT1 cells resulted in not only the anticipated band of the Bcl2-β mRNA variant at the top but also a smaller band of about 500 bps (arrowhead). B: Purification, cloning and sequencing of the smaller band (indicated by the arrow in A) reveal that it is a Bcl2-Nek9 chimera (with the lowercase “t” and “a” at both ends appended by Taq DNA polymerase). The Bcl2-R2i primer (underlined region inside the sequence) has 12 nts (shaded) matched to the Nek9 sequence. There is a 5-nt unmatchable sequence (shaded 5’ACAGA3’ in boldface) after this BCl2-R2i primer. This chimera ends with another Bcl2-R2i sequence (underlined 20 nts in boldface at the 3’ end), but the Bcl2-L1211 forward primer is not in the cDNA.
Fig 8.
Part of the cDNAs cloned via a primer annealing only at 4 or 5 nts during PCR.
Top panel: a cDNA was inadvertently cloned because the last 4 nts, i.e. 5’GGAC3’ (shaded), of our mtR3071 primer (underlined and boldfaced) are reversely complementary and thus annealed to the 2263-2266th bps of the mtDNA (AY195786.2). The lowercase letter “t” at the 5’ side of the mtR3071 sequence was appended by Taq DNA polymerase, and the sequence before this “t” belongs to the T-A vector. Bottom panel: A cDNA was accidently cloned because the last 5 nts, i.e. 5’TCTTC3’ (shaded) in the mtR2694 primer (underlined and boldfaced), is reversely complementary and thus mistakenly annealed to the 3550-3554th-bp region of the mtDNA (AY195786.2). The lowercase letter “a” after the primer was appended by Taq DNA polymerase, whereas the sequence after the “a” belongs to the T-A vector. (“…” sequence omitted)