8 Molecular Anthropology

Ms. Nilupher and Meenal Dhall

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Contents:

    1.      Introduction

2.      Ancient DNA studies

2.1 Authenticity of ancient DNA

3.      Neanderthal – modern human relationships

4.      Molecular markers anthropological genetic studies

4.1 Autosomal marker

4.2 Y- Chromosome DNA marker

4.3 Mitochondrial DNA marker

5.      Molecular techniques

5.1 DNA extraction

5.2 DNA-DNA hybridization

5.3 Polymerase chain reaction

6.      Human genome project

Summary

 

Learning objectives:

1.      It focuses on the study of molecular anthropology.

2.      It enables to understand the importance of ancient DNA studies.

3.      It helps to learn the various molecular markers and techniques use in anthropological genetic studies.

 

1. Introduction

 

The contribution of genetics to biological anthropology has been since a very long time. Many areas of biological anthropology are influenced by genetic processes either directly or indirectly. Further genetic data also provides a clue to biological relationships among individuals or populations. Since the knowledge of the overall genetic constitution or genome of primate species has been increasing, biological anthropology can use genetics in further research as it has not used genetic data earlier. The advance techniques of molecular genetics can make the subject matter of biological anthropology more deeply rooted. First, genetic variation within or between species is now probable in more points. Next, biological anthropologist can also analyse those genes which maintain different range of phenotypic characters instead of confining to the studies of blood groups and proteins. And lastly, they may start to explore morphological adaptation and evolution of primate on molecular basis, and those methods that are being developed now to study the significant variation in anatomy and physiology on molecular basis (Rogers, 1992).

 

Biological anthropology considers the study of adaptive evolutionary change to be a major part. To investigate about the genes which effect the variations between the forelimbs of gibbons, baboons and tarsiers as well as other anatomical system in other species is an early step. Research in the field of molecular variability can be taken an important role by bio-anthropologists. They can investigate the variations in the genetic loci by correlating observed differences with observed morphological differences.

 

The term molecular anthropology was first introduced by an American biologist of Austrian origin, Emil Zuckerkandl. The emergence of Molecular Anthropology can be traced back to the 1962 symposium ‘’Classification and Human Evolution’’ at Burg Wartenstein in Austria. Molecular anthropology is defined as the study of primate phylogeny and human evolution through the genetic information decoded by proteins and polynucleotides (Sommer, 2008). This discipline has not only focussed into the molecule which encodes the genetic information, deoxyribonucleic acid (DNA) since last fifty years, but it has also stretched its application beyond the aspects initially used by Zuckerkandl. For this reason, molecular anthropology is growing as one of the most likely and rapidly advancing sub-fields of Anthropology.

 

Molecular anthropology is a very rapid and ever-growing branch of anthropology which the study of human genetic polymorphisms and holds a great promise for both past and future. Some anthropologists consider the subject, genetic or molecular anthropology as a science of future, however, it must be emphasised as a science of the past as well as present. An extensive advance in the knowledge of diverse key features of human evolution is achieved by rooting into several important issues that have been attained by using a molecular approach. One of the most famous applications of Molecular Anthropology is the pioneering study on mitochondrial variation in worldwide populations by Rebecca Cann and co-workers in the late eighties (Cann, 1987). It has become renowned due to its important inferences for understanding the origin and diffusion of modern Homo sapiens anatomically.

 

2. Ancient DNA Studies

 

Higuchi and his colleagues first demonstrated the study of DNA in ancient specimens in nonhuman material in 1984 (Higuchi et al., 1984). Ancient DNA studies have made possible the investigation of extinct species and populations and they also provided a means of directly tracing the temporary genetic changes (Leonard et al., 2000). However, ancient DNA studies are very difficult to carry out because generation of sufficient authentic data is required. Prior to the advancement of molecular techniques in biology, the study of evolution depend either on traditional analyses of the structural or functional features of living and fossil organisms or on the relevant molecular data from extinct species to the geological past. But evolutionary biologists had tried to make more advances on these studies for many years with molecular information obtained directly from extinct organisms by means of biochemical methods. The techniques of amino acid sequencing applied on the leftovers of proteins in fossils had been proven ineffective. Thus the attention had changed to immunological methods such as radioimmunoassay in order to distinguish tiny quantities of protein remained in bones, skin, and teeth of fossils and on the preserved specimens of museum. After the first successful recovery of DNA from a small piece of 140 years old quagga, an extinct animal of the horse family by Wilson’s group in the year 1984. In the next, an additional report was appeared on the cloning of ancient human DNA of a pre-dynastic Egyptian mummy by Paabo (Rourke et al., 2000). The simultaneous development of the advance molecular technique, polymerase chain reaction had begun research on extraction and characterization of DNA from geographically isolated human samples (Mullis & Faloona, 1987; Saiki et al., 1988).

Figure1: Ancient DNA

 

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2.1: Authenticity of ancient DNA

 

Authenticity of DNA is a principal concern before starting a research work in order to get good results which reveal endogenous target sequences rather than modern contaminants (Handt et al., 1994; Richards et al., 1995). Six criteria had been recommended by Handt and his co-workers (1994) for estimating authenticity of DNA results:

 

(a)  Pre- and post-PCR activities should be consciously separated in the lab, or implemented in different laboratories.

(b)  Laboratory protocols should be strictly adopted and followed to inhibit and monitor the introduction of modern DNA.

(c)  For identifying contamination, controls should be used regularly.

(d)  Confirmation of initial results should be made by using replicate samples.

(e)  Observation of a DNA sequence data should be done to justify phylogenetic sense.

(f ) An inverse relationship between fragment size and PCR efficiency should be observed.

 

3. Neanderthal – Modern Human Relationships

 

Homo neanderthalensis or Neanderthals were archaic hominids considered to be most akin to modern humans. These extinct members of the Homo genus were found to be inhabited in Europe and parts of western and central Asia before their disappearance 25,000 years ago (Tattersall, 1995). It was suggested that Neanderthals were perhaps cohabited with Cro-Magnon man (anatomically modern humans) for 20,000 years (Finlayson et al., 2006). The evidence of simultaneous cohabitation was not found at any single archaeological site. However, their long period of coexistence could have the probability of genetic admixture between Neanderthals and modern humans. The evidence of gene flow between Neanderthals and modern Europeans could be proven by their shared morphological features (Wolpoff et al., 2001; Voight, 2006). In the late 1980s, new developments in molecular technique i.e. fossil-preserved ancient DNA analysis arose in the late 1980s, the relationship between Neanderthals and modern humans had been given more well with genetic evidence and they were believed to be cohabited in Europe for 6000 years and might have the possibility of interbreeding (Krings et al., 1997; Mellars, 2004). The possibility for divergence of lineages of modern humans and Neanderthals in Africa from 300,000 – 700,000 years ago was established by comparisons on both genomic and mitochondrial sequence (Krings et al., 1997). Thus, there was a continuation in comparative studies on modern human and Neanderthal man later on.

 

It is very complicated to analyse human and Neanderthal polymorphism. The splitting of human– Neanderthal was expected to be due to existence many human polymorphisms and thus also might be present in Neanderthal as well. As a result, the fixed replacements in modern humans might be polymorphic in Neanderthal and also both of them might be consequential relative to chimpanzee. Currently there is no clear clue of the effects of Neanderthal polymorphism on substitution maps to be addressed. Genetic diversity of Neanderthal will not be available for some duration and also will be limited to a few individuals even if there is wide insight into genome of Neanderthal. The genome sequence of Neanderthal might help in detecting occurrences of recent positive selection in the genome by identifying the remarkably long runs of association of human specific derived substitutions because of selective sweeps which has occurred early in modern human evolution (Noonan, 2010).

 

4. Molecular Markers in Anthropological Genetics Studies

 

The DNA in human comprises of 22 pairs of autosomes and a pair of sex chromosomes i.e. XX or XY and it is also found outside the nucleus in the form of mitochondrial DNA. Nuclear DNA can be divided into two regions on the basis of function namely, coding and non – coding regions. Human genome carries various types of polymorphisms. Single nucleotide polymorphism (SNP) is the most basic type of those polymorphisms that substitute one base for another.

 

4.1: Autosomal markers

 

The history of a population, population structure, events such as migration and gene flow with the time period of its occurrence are investigated by anthropological genetic studies through the use of molecular markers to characterize different polymorphisms occur at various locations all over the genome. Use of autosomal markers in the studies are more advantageous than those of mitochondrial DNA (mtDNA) markers and Y chromosome present in that because they sample a larger portion of the gene pool which can represent a population as a whole. Autosomal polymorphisms are not restricted to either the paternal or the maternal side because they are transmitted from both the parents and further can provide information about both sexes. On passing down to each generation, recombination and separation of alleles at different loci may be done independently of one another. The human 1.2 (1) collagen gene (COLlA2) is one such example of an autosomal coding marker which was found to be helpful for human phylogenetic research.

 

4.2: Y – chromosome DNA markers

 

Huge amounts of chromatin and few genes that are related to male sex determination are constituents of the Y chromosome. This chromosome is passed solely from father to son. The recombination of this chromosome with the X, other sex chromosomes takes place at only a small portion i.e. the pseudo-autosomal region which is located at the tips of its chromosomal arms. The targeted region of the Y – chromosome for population studies is the portion that does not recombine with other chromosome, referred to as the MSY (male-specific region of the Y). As the MSY does not share a corresponding region on the X chromosome, so it is present in only one copy and it is haploid. The phylogenetic reconstruction of populations is benefitted by Y – chromosome polymorphism as those mutations that have occurred will be passed on to future generations in the absence of recombination. Therefore, it is easy to trace back and identify long-lasting patrilineages to a common male ancestor. Binary markers and STRs are the most commonly used MSY polymorphisms for phylogenetic studies. The classification of new Y chromosomal markers in populations over the world has contributed to their increasing effectiveness for anthropological genetic studies, which until recently lagged behind mtDNA studies.

 

4.3: Mitochondrial DNA markers

 

The other non-recombining portion of the human genome is the mitochondrial DNA (mtDNA). The structure of mtDNA is a double-stranded, circular molecule and located in the mitochondria of the cell which is outside the nucleus (see Figure 2). The mtDNA is inherited from a mother to all of her children i.e. maternally transmitted so only her daughters will be able to pass it again on to successive generations. This marker is subjected to genetic drift and has an effective population size equal to one-fourth that of the autosomes. The copies of mtDNA are present in multiple numbers per cell, hundreds to thousands, depending on the tissue. This feature provides an insight in ancient DNA studies.

Figure 2: Mitochondrial DNA

 

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5. Molecular Techniques

 

With the advancement of technology, many molecular techniques have been introduced in research studies. Those techniques are very interesting as well as hard to carry out. Well knowledge of the techniques is of immense in order to get successful results.

 

5.1: DNA extraction

 

DNA is found in various sources but the best source is considered to be white blood cells. Human blood contains red blood cells, white blood cells and platelets. So in order to extract DNA, separation of different components of blood is important. The blood in the container is treated to prevent clotting and then centrifuge at extremely high speed to settle down the RBC and WBC. The plasma will remain at the top of the container which will be discarded for further steps. After that, white cells can be separated out by adding RBC lysis buffer and centrifuge it repeatedly. To break down the fatty cell membrane, the pellet is further mixed with buffer, soapy, saline solution and to split the cells containing nuclei. The nuclei are again targeted to be broken up by treating it with more soapy solution in order to dissolve nuclear membrane.

Figure 3: Centrifuge machine

 

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The DNA will be dissolved when a high salt concentration solution is added to the mixture. When chill ethanol is added to the mixture containing dissolved DNA, the pool of DNA (Figure 4) will be precipitated out and will be visible to naked eyes as thread like structures of white colour. The pool of DNA will then be transferred to a sterilised tube and containing buffer. This DNA can be preserved for many years at very low temperature for future use.

 

Figure 4: Precipitated DNA

 

(Source Link: www.vivo.colostate.edu)

 

5.2: DNA-DNA Hybridization

 

DNA- DNA hybridization is a technique use to remove repetitive DNA because large segments of repetitive DNA are present in the human and non- human genomes and those sequences are rapidly re-associates mostly after denaturation. The degree of genetic distance between the species is possible to determine when mixtures from different species are formed. The hetero-duplexes which contain the largest number of nucleotide mismatches will disassociate soon. The genetic difference between two species can be estimated by subtracting the difference in temperature at which hetero-duplexes denature from the temperature at which homo-duplexes denature.

 

5.3: Polymerase chain reaction

 

PCR or polymerase chain reaction is a technique use for DNA replication. It has three steps i.e. denaturation, annealing and elongation. Polymerase, an enzyme makes two new strands after all the components of the PCR reaction mixture are together in one tube and the original template is melted which doubles the amount of DNA present. This process is repeated 20 to 40 times in order to yield two new templates in each cycle for the next cycle. This polymerase chain reaction provides an enormously delicate means of amplifying small quantities of DNA.

 

6. Human Genome Project

 

The Human Genome Project (HGP) has been started since late 1970s and early 1980s. During those times highly conceptual and technical advances has been initiated in this field. With this initiation, many difficulties in human molecular biology can be studied in more innovative ways and with great precision. The origins of the HGP can be traced to many fundamental scientific advances in different fields such as molecular biology, genetics and biotechnology. The Human Genome project is the combine effort of the world to plot and sequence the human genome. Various fields of biological and technical researches have associated their skills and therefore try to establish deeper interactions between scientific disciplines. The combine efforts of all these skills should arouse many advances in both pure and applied fields of research and should initiate new and interdisciplinary training programs.

 

The HGP represents an emergence of these fields on the molecular biology of Homo sapiens too. The NIH factsheets (2013) reported that the Human Genome Project has already discovered more than 1,800 disease genes currently. Because of the result of the HGP, many researchers can find a gene suspected of causing an inherited disease in a very few days. About more than 2,000 genetic tests are available for human conditions that enable patients to learn their genetic risks for disease thereby helping healthcare professionals to diagnose disease. The development of haplotype map (HapMap) in 2005 was one of the major steps toward such widespread understanding all over the world.

 

Summary

 

Molecular anthropology plays an important role in the present scenario all over the world. It merges into different fields of genetics and molecular biology to understand and betterment of the human species. Many new ideas and techniques of molecular biology have been introduced in the field of human studies to trace back the origin. The genetic variation among the species can also be established. Neanderthal – human relationship has been estimated through ancient DNA studies from fossil finds. Keys of human evolution have been achieved through the knowledge of diverse advance techniques of molecular techniques. Anthropological genetic studies such as human variation, gene flow, genetic distance are all acquired with the help of molecular markers such as autosomal markers, Y – chromosome DNA markers, mitochondrial DNA markers etc. Thus advance molecular techniques such as DNA- DNA hybridization, polymerase chain reaction and many other techniques help to estimate the genetic differentiation between two species. Human genome project is making a combined efforts with other fields of science or making the present and future generations better than that of the past life.

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