# Fragment of the AncientRbcL Gene from the Miocene

Authors: Patricia Veiga-Crespo; Lidia Ruiz Martinez; Miguel Viñas; Tomás G. Villa
Journal: Scholarly Research Exchange
 (2008)

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## Abstract

The development of molecular biology techniques has allowed a new approach to palaeontology and studies on ancient DNA. As a plant fossil resin, amber provided a good matrix for preserving ancient biological material. Some difficulties arise when experimental work is done to extract information concerning these preserved specimens. The major risks in this type of works
are the contamination with modern DNA and the degradation of the
ancient DNA. A safe method to sterilize amber stones has been designed allowing the amplification of a fragment of the ancientRbcL gene from the Miocene (c.a. 25 million years). Presumably, the gene was from Hymenaea protera, an extinct member of the Leguminoseae family. The phylogenetic tree and divergence rates indicate that since although it is a well-conserved gene, and then should be a good candidate for studying the evolution of plant macrogroups, probably it is not good enough for analyzing divergence among closely related species.


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## Body

## 1. Introduction

The development of PCR-based techniques has prompted
studies on fossil DNA as well as in molecular palaeontology. In the pursuit of
such goals, a crucial issue is the availability of a good supply of
well-preserved ancient DNA, amber being one of the best such sources because of
its high capacity of specimen preservation.This material is formed through the fossilization of resins
secreted by conifers and angiosperms [1]. The amber from Baltic was formed
during the Oligocene from the members of conifers mainly (c.a. 40–50 million years)
[2]. The amber from the Dominican Republic
was mainly formed with resin from the Leguminoseae during the Miocene (c.a. 25 million years), mainly from
members of the genus Hymenaea (i.e., Hymenaea protera), an extinct member of
the Leguminoseae family.There are two areas with major amber deposits in the Dominican Republic: the Northern area (Santiago
de los Caballeros) and the Eastern area (Cordillera
Oriental) (Figure1(a)). Paleogeographically, both areas form part
of the same sedimentary basin, which was disrupted by movements along major
faults (Figure 1(b)).(a) Map of the amber mining in Dominican Republic. (b) Stratigraphic
layers of amber deposits.
(a)(b)During the amber-forming process, a variety of samples,
including insects, pollen, and microorganisms, became embedded in the resin and remained unaltered for millions of years
despite deleterious external agents as water or ultraviolet radiations
[3–5].The current
problems involved in working with this type of ancient DNA are contamination by
contemporaneous DNA, the occurrence—in the sample—of
PCR-inhibiting agents [6], and the degradation of the DNA itself in the amber
[7, 8]. The prevention of such
contamination is crucial in this type of studies and must be carried out via an
appropriate sterilization procedure that is sufficiently aggressive to
sterilize the surface of the amber and yet mild enough to preserve the ancient
DNA.Choice of the ancestral genes to be studied is a critical
point in the outcome of these studies, and, in the present work, we decided to
use oligonucleotides able to amplifyRbcL gene (RuBisCo large subunit), an essential and highly conserved gene from
plants.


## 2. Material and Methods

### 2.1. Amber Samples

The present work was carried out using samples from amber
nuggets from Santiago de Los Caballeros Mountain (Dominican Republic), dated
stratigraphically as 15–30 million years
old [9].


### 2.2. Sterilization Procedure

This was basically according to Lambert et al. [10], with some modifications
described by Veiga-Crespo et al. [9]. The incubation time in 2% glutaraldehyde (Merck, USA) at
40°C was divided into two 24 hours periods, and after the first period the
solution was changed. The ultrasound treatments (Ultrasons P, Selecta, Spain) were for 30 minutes
and
this ultrasonic procedure was implemented throughout the sterilization procedure
(24 hours period in 10% calcium chloride at 25°C and 24 hours period in 70%
ethanol at room temperature) being interrupted before changing the amber
nuggets to brain heart infusion broth medium (B.H.I.B.; Biolife).


### 2.3. Fracture of Nuggets

Before fracturing,
the samples were kept for fifteen days at 21°C, fifteen days at 30°C and
fifteen additional days at 37°C in B.H.I.B. in order to ensure a lack of
microbial growth at the three temperatures. The amber was then frozen in liquid
nitrogen and ground in a mortar. The powder was resuspended in B.H.I.B. and the
aliquots kept at −70°C until use.


### 2.4. Extraction of Fossil DNA

Extraction of fossil DNA was accomplished using the Ancient
DNA kit (GeneClean, Bio 101).


### 2.5. PCR Reactions and PCR-Products Purification

Oligonucleotide design was done according to the current
sequence of theRbcL gene from Pinus edulis (Genbank accession code:
X58137). The oligonucleotides used were RbcL-forward:
5′-ATGTCACCAAAAACAGAGAC-3′ and RbcL-reverse:
5′-ATGTCACCAAAAACAGAGAC-3′.The PCR mixture was 1 U Taq polymerase (Takara Shuto Co.),
2 ng/μL BSA (Promega), 0.5 μM of each oligonucleotide (Invitrogen),
2 mM MgCl2 (Takara Shuto Co., Japan),
0.2 mM dNTPs mix (Takara Shuto Co.), and deionized sterile water to a final
volume of 50 μl. The reaction was accomplished in a Robocycler Gradient 96
device (Stratagene)
with the following program: 1 cycle of 5 minutes at 94°C; 35 cycles of 1 minute
at 94°C, 30 seconds at 56°C and 1 minute at 72°C. The process was ended with a
cycle of 10 minutes at 72°C.The PCR-products were purified using Wizard PCR preps kit (Promega, USA). DNA sequencing
was performed according to Sanger's method [11], employing duplex DNA and using the T7
Sequenase v2.0 PCR Product sequencing kit (Amersham Pharmacia Biotech, Spain),
following the manufacturer's instructions. Labelling was done with Redivue
[35S]-dATP.


### 2.6. Sequence Analyses

Sequence analyses and determination of phylogenetic
relationships were performed using the ClustalW application, included in the
software VectorNTI Advance Suite v9.0 (Informax). The distance matrixes were
performed using the Biology WorkBench 3.2 from San Diego Supercomputer Center,
employed the ClustalW algorithm [12] and
the PHYLIP algorithm [13].


### 2.7. Controls of Contamination

Working surfaces were periodically treated with ethanol
(70%) and before and after each work session the nuggets were treated with 10%
sodium hypochlorite (Merck). All culture media were maintained for 15 days at
21, 30, and 37°C before use. All solutions used to sterilize the amber stone
surfaces were previously filtered through 0.22 μm membranes (three times) that
had been previously sterilized. Before stone grinding, the samples were
incubated in B.H.I.B. medium and subjected to the same temperature cycle in order
to discard any possible contamination. After grinding, microbial contamination
was investigated again. The solutions used for DNA extraction and PCR were
periodically controlled for fortuitous contamination. Throughout the process,
particular care was taken in order not to use
glassware or equipment that had been previously in contact with current DNA.


### 2.8. Criteria of Sequence Validity

The following criteria
were adopted (i) DNA was extracted only from stones that had passed all the
contamination checks; (ii) samples from the same stones had to show similar
results; (iii) ancestral sequences had to show homology with current ones and
had to display phylogenetic coherence, and (iv) large DNA fragments (longer
than 1 Kb) had to be discarded to avoid either sample contamination with
current DNA or jumping-PCR phenomena.


## 2.1. Amber Samples

The present work was carried out using samples from amber
nuggets from Santiago de Los Caballeros Mountain (Dominican Republic), dated
stratigraphically as 15–30 million years
old [9].


## 2.2. Sterilization Procedure

This was basically according to Lambert et al. [10], with some modifications
described by Veiga-Crespo et al. [9]. The incubation time in 2% glutaraldehyde (Merck, USA) at
40°C was divided into two 24 hours periods, and after the first period the
solution was changed. The ultrasound treatments (Ultrasons P, Selecta, Spain) were for 30 minutes
and
this ultrasonic procedure was implemented throughout the sterilization procedure
(24 hours period in 10% calcium chloride at 25°C and 24 hours period in 70%
ethanol at room temperature) being interrupted before changing the amber
nuggets to brain heart infusion broth medium (B.H.I.B.; Biolife).


## 2.3. Fracture of Nuggets

Before fracturing,
the samples were kept for fifteen days at 21°C, fifteen days at 30°C and
fifteen additional days at 37°C in B.H.I.B. in order to ensure a lack of
microbial growth at the three temperatures. The amber was then frozen in liquid
nitrogen and ground in a mortar. The powder was resuspended in B.H.I.B. and the
aliquots kept at −70°C until use.


## 2.4. Extraction of Fossil DNA

Extraction of fossil DNA was accomplished using the Ancient
DNA kit (GeneClean, Bio 101).


## 2.5. PCR Reactions and PCR-Products Purification

Oligonucleotide design was done according to the current
sequence of theRbcL gene from Pinus edulis (Genbank accession code:
X58137). The oligonucleotides used were RbcL-forward:
5′-ATGTCACCAAAAACAGAGAC-3′ and RbcL-reverse:
5′-ATGTCACCAAAAACAGAGAC-3′.The PCR mixture was 1 U Taq polymerase (Takara Shuto Co.),
2 ng/μL BSA (Promega), 0.5 μM of each oligonucleotide (Invitrogen),
2 mM MgCl2 (Takara Shuto Co., Japan),
0.2 mM dNTPs mix (Takara Shuto Co.), and deionized sterile water to a final
volume of 50 μl. The reaction was accomplished in a Robocycler Gradient 96
device (Stratagene)
with the following program: 1 cycle of 5 minutes at 94°C; 35 cycles of 1 minute
at 94°C, 30 seconds at 56°C and 1 minute at 72°C. The process was ended with a
cycle of 10 minutes at 72°C.The PCR-products were purified using Wizard PCR preps kit (Promega, USA). DNA sequencing
was performed according to Sanger's method [11], employing duplex DNA and using the T7
Sequenase v2.0 PCR Product sequencing kit (Amersham Pharmacia Biotech, Spain),
following the manufacturer's instructions. Labelling was done with Redivue
[35S]-dATP.


## 2.6. Sequence Analyses

Sequence analyses and determination of phylogenetic
relationships were performed using the ClustalW application, included in the
software VectorNTI Advance Suite v9.0 (Informax). The distance matrixes were
performed using the Biology WorkBench 3.2 from San Diego Supercomputer Center,
employed the ClustalW algorithm [12] and
the PHYLIP algorithm [13].


## 2.7. Controls of Contamination

Working surfaces were periodically treated with ethanol
(70%) and before and after each work session the nuggets were treated with 10%
sodium hypochlorite (Merck). All culture media were maintained for 15 days at
21, 30, and 37°C before use. All solutions used to sterilize the amber stone
surfaces were previously filtered through 0.22 μm membranes (three times) that
had been previously sterilized. Before stone grinding, the samples were
incubated in B.H.I.B. medium and subjected to the same temperature cycle in order
to discard any possible contamination. After grinding, microbial contamination
was investigated again. The solutions used for DNA extraction and PCR were
periodically controlled for fortuitous contamination. Throughout the process,
particular care was taken in order not to use
glassware or equipment that had been previously in contact with current DNA.


## 2.8. Criteria of Sequence Validity

The following criteria
were adopted (i) DNA was extracted only from stones that had passed all the
contamination checks; (ii) samples from the same stones had to show similar
results; (iii) ancestral sequences had to show homology with current ones and
had to display phylogenetic coherence, and (iv) large DNA fragments (longer
than 1 Kb) had to be discarded to avoid either sample contamination with
current DNA or jumping-PCR phenomena.


## 3. Results and Discussion

Because of the size of the gene and the state of
degradation of the ancient DNA, amplification of the complete gene from the
samples was unsuccessful. It was, therefore, necessary to design internal
oligonucleotides from the initial region of the gene.Amplification of a segment of the ancientRbcL gene was positive in Miocene stones
from the Dominican Republic but negative in Oligocene amber samples (c.a. 40 million years old), which were
positive for other genes [9, 14]. A consensus sequence was elaborated (Genebank
accession code: AY484432) from all the ancient DNA sequences. This Miocene
sequence showed 82% similarity to the current Pinus edulis RbcL gene
and it was by far the most highly conserved sequence in all the genes found in
both Miocene and Oligocene stones [9, 14].When the obtained consensus sequences were compared with
the actual ones ofP. edulis, it was
seen that 1% of the total changes were insertions or deletions (22% and 78%,
resp.) and the rest corresponded to transversions or transitions (42% and 58%,
resp.). As a result of all these changes,
the actual gene fragment is thirteen nucleotides shorter than the miocenic one;
it would be worth trying to corroborate this tendency to shorten gene size
through cloning of new RbcL fragments.The new amplified fragment was 122 pb longer than the
previous one reported by Poinar Jr.
[15] for Hymeneaea protera sp. n. (GenBank DataAccess: L08477). When both fragments were compared with each other, it
was seen that 15% of the total changes were insertions or deletions whereas the
rest corresponded to transversions or transitions.When the three sequences were aligned (Figure2) and the
distance matrix elaborated, it was observed that the both miocenic sequences
were closer than actual P. edulis one. These distance values were observed independently of the algorithm used
for calculation the distance (Table 1).Table 1
Distance matrixes ofP. edulis, H. protera sp. n.
and miocenic sequences. (a): by ClustalW
algortithm; (b): by Phylip algorithm.
(a)H._proteraThis_workP.edulisH._protera0.0000.1540.182This_work0.1540.00000.154P.edulis0.1820.1620.0000(b)H._proteraThis_workP.edulisH._protera0.00000.18060.2127This_work0.18060.00000.1864P.edulis0.21270.18640.0000Figure 2
Alignment between the three sequences.Next, a phylogenetic tree was drawn according to the data
in GenBank (Figure3). Phylogenetic analysis was done using the neighbor-joining
method with the lowest possible evolutionary events. Ambigous residues were
resolved as “gaps” and transversion/transition substitutions were given more
importance than insertion/deletion events.Figure 3
Phylogenetic tree ofRbcL gene.TheRbcL gene is
highly conserved among plants and, as it is subjected to high selective
pressure, it shows a low degree of evolutionary divergence; it is thus a good
candidate for fossil DNA work. In the phylogenetic tree, it may be seen that in
general the different groups do appear correctly located except for the
sequence of Pinus halepensis, which
is located at an algal junction. This analysis again confirms the position of
the amplicon in the group formed by the actual and extinct members of genus Hymeneaea and it keeps it away from the
actual sequences belonging to the conifer members despite the fact that these
latter sequences were employed for oligonucleotide design and the high
conservation of RbcL gene.The fact that our Miocene sequence was found among dicot
plants instead of among gymnosperms may be attributed to the abovementioned
highly conserved sequence, although it should be borne in mind that dicots were
the precursors of monocots, either through syncotylic
events or through heterocotylic events after a chain of progressive
anisocotylic events [16].Since the Miocene sequence and that ofPinus edulis (used for oligonucleotide synthesis) exhibited 82% of
homology and the highest conserved grade between all the species studied in
this work, we suggest that this gene may be useful for studying
macroevolutionary lines in plants, although it may not be as suitable for
studies of divergence and evolutionary rates among closely related species.



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*Source: 417592-2008-11-16.xml*