The Case of Lactase Persistence Evolution in Humans

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Introduction These slides are provided as a teaching resource for the Lactase Persistence case as described on www. evo-ed. com. A fuller description of the case can be found on the website. Teaching notes can be found in the notes section beneath each slide when viewing the slides in “Normal View” in Power. Point. To select this option in Power. Point, go to the main menu, choose “View” and then “Normal. ”

The Cell Biology of Lactase Persistence

Biology of the Digestive Tract Enterocytes (the cells that line the inside of digestive tract) are responsible for breaking down and absorbing nutrients from the food in the stomach and small intestine.

First Food: Mother’s Milk The enterocytes of all infant mammals exhibit high levels of lactase during infancy, when milk is the main source of nutrition. Photo credit: Jim French, Flickr

Lactase Unlocks an Energy Source Lactose is a disaccharide sugar found in milk. Lactase breaks down lactose into two monosaccharides, glucose and galactose. These simple sugars can be absorbed by cells in the small intestine and used as a source of energy.

Location of Lactase This is the “brush border” of an enterocyte, which is the side of the enterocyte that comes in contact with the contents of the small intestine. Lactase is stained brown.

Lactase Breaks Down Lactose

Glucose and Galactose are Absorbed INTESTINE CONTENTS ENTEROCYTE BLOODSTREAM

Lactase Regulation Almost all known mammals – including 65% of humans – experience a decrease in lactase biosynthesis in the years after weaning. The regulation of lactase biosynthesis after weaning is the main factor that separates Lactase Persistent from Lactase Non-Persistent individuals.

Lactase Regulation Why does this happen? Why can’t adult mammals digest milk?

Lactase Regulation The decrease in lactase production after weaning is likely a matter of energy conservation at a cellular level: 1. It takes energy to produce any enzyme, including lactase, the enzyme needed to digest milk. 2. Typically, mammals do not consume milk once they have stopped nursing. 3. Without milk consumption, energy spent producing lactase would be energy wasted at the cellular level. Therefore, over time, the more energetically favorable option has been selected for: a decrease in lactase production after weaning.

Lactase not Produced in Adults What happens to humans then, if they continue to drink milk into adulthood?

Lactase not Produced in Adults If undigested lactose passes into the large intestine, it will trigger the symptoms of Lactose Intolerance. 1. The increased sugar concentration in the large intestine creates an osmotic gradient that draws water into the gut. This causes cramping and diarrhea. 2. Bacteria in the large intestine digest the lactose as food, creating gaseous by-products like methane, carbon dioxide, and hydrogen. This leads to gas build-up and flatulence.

Lactase Persistence HOWEVER… 35% of humans do produce lactase after weaning, and are therefore able to continue to consume milk and other dairy products into adulthood.

Discussion 1. How do you think this is possible? 2. What changed in order for these adult humans to be able to digest milk?

Discussion 1. How do you think this is possible? This is possible through Lactase Persistence, or the continued production of lactase at high levels throughout adulthood. 2. What changed in order for these adult humans to be able to digest milk? The down-regulation of lactase biosynthesis that normally occurs must have been prevented or counteracted.

Review 1. 2. 3. 4. What are enterocytes? Where is the lactase enzyme located? What does lactase do? Why can’t most adult mammals drink milk? a. What happens if they do? 5. Why is lactase regulated?

Molecular Genetics Lactase Persistence

The Lactase Gene • The Lactase gene is ~55, 000 base pairs in length. It is translated into a protein, Lactase, that is 1927 amino acids in length (shown below). MELSWHVVFI ASQITHYKVF SFGDLVGIWF DTVDFLSLDL SCSSSSKKSM WAEGGRGVSI IDRLQDAGIE SDPGVASFKV YPATLRTQIQ SSSWIRVVPW LIDGFEGPSG KVVWEKFSSQ ADLNMLRALK LFDSYADFCF SLSLSTHWAE ADVFCLNTYY PNTEDTDRIF NNGMPLARED NLGVSHYRFS VLFQRLGDKV DWAEPRDPSN NHYTTVLAYN NDTARIYYLR PDPATGPHAC LSPVSSF ALLSFSCWGS LSWAQLLPAG TFSDLEEVIK SYECQNEASL SCSLTGSLAL WDPRRPLNTT PMATLFHWDL AHLVLKAHAR QMNRQCSHPV GIRRLLQFVS YSQRFGLHHV PKFERDLFYH VKAYRFSISW QTFGDRVKFW PKSPGVPRDV SRIVQHKTPR YHKTYINEAL EFLYGRFPEG ISWSRILPDG KFWITLNEPF QEDVEAARRY LNYATAISSF TYINEALKAV LHQPDAGPTI DWESDRNFIS STQNPDEKTV ELPHQESRAS RQKLSKLQTI QPDQQQDHET EGQATLEVAS PQALQDHGGW TWHHYNSHHR AQLPEFTEAE LEYTRGKVPI NFSDSSKSRT GTFRDDFLWG SRIFPTGRNS MTFNEPMYLA EAADRMLQFS LNPPSYEDDQ KAYRLDGIDL FIWSAASAAY TTRYINEAGL VIAYQGYGYG VQFMGGWFAH DADRGVASIA QDKVDLRGYT SPVRQEEVQF TAGPLTNDLL QCYRRLLKAL QLQTLSDAHR EPKVKVFIFN TDSSPASAYQ DSYHKVASDV QNESVVDAFL PQQQGHVGIV KQLLKGSADF YLAGNGMPIG PRKSAYFFTS VSSSAYQIEG SINSHGVDYY WLGYGSGEFP LGWFAHPIFR EMAEEEDPSW RGYVAWSLMD QIEGAWRADG NYYVRLIDTL TAAPGVSNRP PIFKNGDYNE DRSWPDSGSF VWSAMDNFEW LGLMLGTTEA HNLSGLLGDQ KTARLQPMVI KAYEIYHESY LKLPDCPSTM RIWEAFANQS ALLCGLRAQV DYAAFCFSTF LNSDWAEPLS LGLSHYTSRL ESENLFDDSL IIEKNGFLTK AWDADGKGPS NRLINGLVAS PGVKDPGWAP NGDYPDTMKW PSTAMNRAAP NFEWLNGYTV KGLSIWDTFS LAASIQPQVT GTAPYIVGHN VMKTRIRDRS WLKMTPFGFR ATGFSERFGL QTALYVLFSL SSNFVAGDKD LHHQTLPAST AFQGGKLSVV KNPASLLFSL RAERDAFLQD YKFSISWSRI GDRVKLWVTF PERPEDLRAS ISNAPQNTCI RVDYFNQYIN GAKRLLPPNT IWDNFTHTPG NIFPMVTLFH YRIAHAVIKA KVGNRSELQH WGTRRLLNWI KFGLYHVDFN HTPLRVENDA IYHWDLPQTL LIKAHAEAWH LAAGLNKSRL RILNWLKEEY HFVNYSDPSL VLLGVCGLAF MYVCHQPLPT LRRTEAFADL LRAEDIPELL FEAINKDQVL TFPEGFLWGA FPMGHGSSPS HEPWVMSYAG ERFLHFMLGW PSYDTIGGFS EVLKAIKEDS VNLPSKVRAF SNVKDNATGD WDLPQALQDI HARVYHTYDE LATSRLPSFT KEEYGDIPIY NTNRPRTARA IGDVACDSYH QDVGGWENET LYNDVYRASQ PEFTESEKRR NDPPIYVTEN PRIPKASAKF LSYKYCKRSK FLPEYFSSLH FADYATFAFH LEPPISALAQ TIGFDINEFL STGAFNVEGG LPGVAYYNKL YGTGQHPPGI FAHPVFVDGD QHVNHVWPQT VDVRSYIARS TFPSEVPSKA IACDSYHQLD GGWENPALID KYRQEQKGVI EEEKRFIRAT ITENGVGLTN SARYYTEVIT KIAEDLVTLQ IVQRFKEYAD GGVISITISS INGTYDFFGF GVSQREETDL YASVVRCNGF QGKTQRSQQE

Lactase Biosynthesis 1. 2. 3. 4. Transcription (controlled by Transcription Factors) Translation Post-Translational Modifications Expression on the Cell Membrane

1. Transcription Within the nucleus, the Lactase gene is transcribed into Lactase-m. RNA by RNA polymerase. Note that other factors, also shown in blue, are important for the function of RNA polymerase. The rate of transcription is controlled by Transcription Factors shown here in red and green.

Transcription Factors A transcription factor (TF) is a protein that binds to a specific segment of DNA and influences the transcription of a gene.

Transcription Factors Once bound to DNA, a Transcription Factor can attract the molecular machinery necessary for transcription. TFs can even attract other transcription factors (and they often do), forming a large transcription complex. We will focus on two different types of TFs, general transcription factors and regulatory transcription factors.

General Transcription Factors General transcription factors, shown as a complex of proteins, bind to Promoter Sites on the DNA, and are responsible for initiating transcription by binding to RNA Polymerase and associated factors, as well as to other regulatory transcription factors.

Regulatory Transcription Factors Regulatory transcription factors can act as activators or repressors. Activators increase expression and repressors decrease expression of a particular gene.

Regulatory Transcription Factors: Activators bind to Enhancer Sites which are particular stretches of DNA, and influence the probability and frequency of transcription by attracting general transcription factors to the transcription complex.

Enhancers Enhancer Sites may be far away from the start of a gene, but this allows Activators to cause large loops in the DNA, which can bring many distant but important transcription factors all together around one large transcription complex. This increases the rate of transcription of the gene.

Why is this important? Because there is a mutation associated with Lactase Persistence that affects the binding ability of Transcription Factors.

What Kind of Mutation is This? A Single Nucleotide Polymorphism, or SNP, is a type of mutation that involves changing one letter in the genetic code. One letter in billions of letters may seem insignificant, but a SNP can affect how strongly (and therefore how often) a General Transcription Factor or Activator will bind to that piece of DNA. This can have very profound effects on the transcription, translation, and expression of genes.

The SNP in Lactase Persistence In lactase persistence an enhancer site located 13910 base pairs upstream from the Lactase gene has a “C” replaced by a “T. ” This SNP increases how strongly and how often the transcription factor Oct 1 binds to this site. As an activator, Oct 1 causes more general transcription factors to bind to the Lactase gene. . . Leading to more transcription of Lactase-m. RNA!

Normal Adult Mammals 1 • After weaning, there is a decrease in transcription factor activity at the Lactase Gene 2 • This leads to decreased transcription of the Lactase gene 3 • The result is lower levels of Lactase in the enterocytes, and the inability to digest lactose in milk

Mutant (Lactase Persistent) * • A mutation at an Enhancer site located 13190 base pairs upstream of the Lactase gene increases binding of an Activator called Oct 1 1 • Oct 1 attracts more general transcription factors to the Lactase gene throughout the individual’s adult life. 2 • This prevents the decreased transcription of the Lactase gene that would normally happen. 3 • The result is steady levels of Lactase in the enterocytes, and a retained ability to digest lactose in milk throughout adulthood.

Notes for Previous Slide The ER and Golgi Apparatus process the polypeptide Pre-Pro-Lactase into its mature form, enzymatic Lactase (a protein). 1. Within the Rough E. R. (Endoplasmic Reticulum): a. The signal sequence, whose purpose it was to attach the ribosome to the ER during translation, is cleaved off after translation. b. The membrane-bound polypeptide, now called Pro-lactase, is then dimerized (attached to another copy of itself. ) 2. Then a transport vesicle containing Pro-lactase blebs off the ER and travels to fuse with the Golgi Body. 3. Within the Golgi Body: a. The “pro” subunit of Pro-Lactase prevents degradation and ensures proper folding of the polypeptide. b. This subunit is then cleaved off, leaving two Lactase polypeptides, which together make a mature Lactase enzyme. 4. A vesicle containing the membrane-bound Lactase blebs off the Golgi… 5. and travels to fuse with the cell membrane, which is the brush border membrane of enterocytes.

2. Translation Lactase-m. RNA is translated by a membrane-bound ribosome into an amino acid called Pre-Pro-lactase. lipid During translation, the polypeptide is fed into the ER (endoplasmic reticulum), but remains anchored in the bilayer of the ER membrane after the ribosome has departed.

3. Post-Translational Modification 1. Rough E. R. 2. Vesicle 3. Golgi Body 4. Vesicle 5. Cell Membrane

Review 1. What is Transcription? 2. What are the different kinds of Transcription Factors, and what do they do? 3. What is a SNP? 4. How does the SNP associated with Lactase Persistence lead to increased transcription of Lactase in adulthood?

Anthropology and Biogeography of Lactase Persistence

The Neolithic Revolution The Neolithic revolution describes a period of time, between 12, 000 and 6, 000 years ago, during which humans around the world began transitioning from a hunter-gatherer lifestyle to a farming-herding lifestyle.

Neolithic Revolution This time period saw a burst of innovation as humans developed new ways to interact with their environment, such as tools for planting and reaping crops, mills for grinding grains, and pottery for storage of food.

Neolithic Revolution There were also conceptual innovations driven by this transition, such as the calendar, and the concepts of property and monetary systems. The ancient Egyptian calendar was divided into three seasons based on agricultural activities.

Pastoralism, the cultural practice of milking livestock (such as goats, sheep, cows, and camels), was another innovation of the Neolithic Revolution. It was adopted in various cultures between 12, 000 and 7, 000 years ago.

Milking Livestock and Drinking that Milk The Biocultural Coevolution theory proposes that pastoralism and lactase persistence coevolved. This means that they arose around the same time*, and both changes were reinforced by each other.

How Did Lactase Persistence and Pastoralism Spread?

Current Distribution of Lactase Persistence Fraction of adults with LP trait

Two Different Histories Lactase persistence and pastoralism arose and spread through Europe and Africa independently, an example of convergent evolution. Convergent Evolution is the independent evolution of similar features in separate lineages. This leads to two different “stories” of how Pastoralism and Lactase Persistence arose.

The Story of LP in Europe The earliest evidence of pastoralism was discovered in the Middle East: bones of young cattle, slaughtered before their first birthdays, indicate that humans in the area had begun domesticating and milking cattle. The cattle bones found in the Middle East were about 10, 500 years old.

Migration The earliest domesticated cattle in Greece and the Balkan States (8, 000 years old) were more closely related to the domesticated cattle from the Middle East than the wild cattle found in Europe at the time. This indicates that migrants from the Middle East brought their cattle with them. 8, 000 years ago 10, 500 years ago

Migration 7, 500 years ago 6, 500 years ago 8, 000 years ago 10, 500 years ago Because Middle Eastern cattle herders had more advanced food technology, they easily out-competed the local hunter-gatherers they encountered in Central and Northern Europe.

An Apparent Paradox? Anthropological evidence places the advent of pastoralism at 10, 500 to 6, 500 years ago. However, genetic research says the lactase persistence trait did not become widespread in Europe until 7, 000 to 5, 000 years ago. This suggests that for several thousand years some humans were milking sheep, goats, cows, or camels despite being unable to digest milk. What was going on?

Cheese It is likely that Neolithic humans fermented milk into cheese, which greatly reduced the lactose content of the dairy product, making it more accessible. Ancient pottery remnants like this, found in Northern Europe, were likely sieves used to strain and ferment milk into cheese

A Brief History of Cheese Many Paleolithic and Neolithic cultures practiced the storage and transport of food and water in animal skins and intestines. When milk was introduced to the Neolithic diet in the Middle East it was likely stored in an inflated cow stomach, resulting in the separation of the curds and whey. Cheese is thought to have accompanied pastoralism since its cultural genesis, and is still very popular in European cultures.

The Story of LP in Africa In Africa, lactase persistence evolved independently from the European lineage. The mutations responsible for lactase persistence are different. Prevalence of Lactase Persistence Prevalence of T-13910 SNP

The Story of LP in Africa Neolithic humans in Africa experienced similar selective pressures to adopt pastoralism and evolve lactase persistence. Through slightly different mutations, G-13915 and G-13907, different peoples in Kenya and Sudan evolved lactase persistence via the same Oct 1 transcription factor enhancer site.

The Story of LP in Africa Notice how close the mutation (SNP) sites for Kenya and Sudan are to the European SNP, T-13910. All of these mutations affect the enhancer site of Oct 1, the activator that increases promoter activity at the Lactase gene.

How Did Lactase Persistence Spread? Lactase persistence is thought to have arisen and spread through two types of natural selection: positive selection (selection for advantageous traits) and negative selection (selection against disadvantageous traits).

How Did Lactase Persistence Spread? Negative Selection Positive Selection (disadvantages of Lactose Intolerance) (advantages of Lactase Persistence) • Individually: Milk supplies protein, • Individually: Non-LP individuals fat, sugar, and vitamins, and is missed out on a potentially dependable despite cold weather important source of nutrition and hydration and/or bad crops • Experiencing painful, dehydrating • Neolithic women who could digest symptoms upon consuming milk were estimated to produce (see slide 15), which could be 32% more offspring. deadly. • Culturally: Milk is a more efficient • Culturally: Without pastoralism, protein source: it does not require herders must slaughter their killing livestock, yet the milk from livestock to gain dietary protein one cow nearly equals the caloric from their meat. value of the meat from a whole cow.

How Did Lactase Persistence Spread? Lactase Persistence is a dominant trait. This is also an important aspect of how LP spread throughout the population over time. “Dominant trait” means that if one parent is homozygous for lactase persistent, all the children will be lactase persistent.

Review 1. When did pastoralism originate in the Middle East? 2. How did pastoralism spread in Europe? 3. Why is cheese an important part of the history of Lactase Persistence? 4. How did Lactase Persistence spread in Africa? 5. What selective pressures drove the evolution of LP?

Advanced Study Questions • What is the Vitamin D/Cold Weather hypothesis concerning the latitude-associated distribution of Lactase Persistence? • There is a culture in Africa with the ability to drink milk without ill effect despite not being lactase persistent. Who are they and how is this possible?