Structure and Analysis of Eukaryotic Genes

Split genes Multigene families Functional analysis of eukaryotic genes

Split genes and introns

The mRNA-coding portion of a gene can be split by DNA sequences that do not encode mature mRNA
• Exons code for mRNA, introns are segments of genes that do not encode mRNA.
• Introns are found in most genes in eukaryotes • Also found in some bacteriophage genes and in some genes in archaea

R-loops can reveal introns
mRNA coding regions (exons) separated (by introns) on the chromosome

Examples of R-loops in mammalian hemoglobin genes

Types of exons

Finding exons with computers
Ab initio computation –
Uses an explicit, sophisticated model of gene structure, splice site properties, etc to predict exons •
Compare cDNA sequence with genomic sequence – BLAST2 alignments between cDNA and genomic sequences

Find exons for HBB

Sequence for the human beta-globin gene (HBB): – Accession number L48217 – Thalassemia variant •
Sequence for HBB mRNA – NM_000518 •
Retrieve those from GenBank at NCBI– Get the files in FASTA format •
Run Genscan and BLAST2 sequences

Genscan analysis of the HBB gene

BLAST2: HBB gene vs. cDNA

Introns are removed by splicing RNA precursors

Alternative splicing can generate multiple polypeptides from a single gene

The mRNA for Protein A is made by splicing together exons 1, 2 and 3:

Alternative splicing can generate multiple polypeptides from a single gene, part 2

Or, by an alternative pathway of splicing that skips over exon2, Protein B can be made:

Multigene families, e.g. encoding hemoglobin

Blot-hybridization analysis showing multiple beta-like globin genes in mammals

Gene Expression: where and how much?

A gene is expressed when a functional product is made from it.
• One wants to know many things about how a gene is expressed, e.g. –In which tissues?
–At what developmental stages?
–In response to which environmental conditions?
–At which stages of the cell cycle?
–How much product is made?

RNA blot-hybridizations = Northern

RNA blot-hybridization: Stage specificity

RT-PCR to detect RNA

In situ hybridization and immunoreactions

Sequence everything, find function later

Determine the sequence of hundreds of thousands of cDNA clones from libraries constructed from many different tissues and stages of development of organisms of interest.
• Initially, the sequences are partials and are referred to as expressed sequence tags (ESTs).
• Use these cDNAs in high-throughput screening and testing, e.g.
expression microarrays (next presentation).
Massively parallel screening of high-density chip arrays
• Once the sequence of an entire genome has been determined, a diagnostic sequence can be generated for all the genes
Synthesize this diagnostic sequence (a tag) for each gene on a high-density array on a chip, e.g. 6000 to 20,000 gene tags per chip.
• Hybridize the chip with labeled cDNA from each of the cellular states being examined.
• Measure the level of hybridization signals from each gene under each state.
• Identify the genes whose expression level differs in each state. The genes are already available.

Expression profiling using microarrays

Find clusters of co-regulated genes

Search the databases

What can be learned from the DNA sequence of a novel gene or polypeptide?
Many metabolic functions are carried out by proteins conserved from bacteria or yeast to humans – one may find a homolog with a known function.
Many sequence motifs are associated with a specific biochemical function (e.g. kinase, ATPase). A match to such a motif identifies a potential class of reactions for the novel polypeptide

Databases, cont’d

One may find a match to other genes with no known function, but their pattern of expression may be known.
Types of databases:
– Whole and partial genomic DNA sequences
– Partial cDNAs from tissues (ESTs
= expressed sequence tags)
– Databases on gene expression – Genetic maps

Express the protein product

Express the protein in large amounts – In bacteria – In mammalian cells – In insect cells (baculovirus vectors) • Purify it
Assay for various enzymatic or other activities, guided by (e.g.) – The way you screened for the clone – Sequence matches
The phenotype of directed mutation
Mutate the gene in the organism of interest, and then test for a phenotype
The gain of function – Over-expression – Ectopic expression (where normally is silent)
Loss of function – Knock-out expression of the endogenous gene (homologous recombination, antisense) – Express dominant-negative alleles – Conditional loss-of-function, e.g. knock-out by recombination only in selected tissues

Localization on a gene map

E.g., use gene-specific probes for in situ hybridizations to mitotic chromosomes.
Align the hybridization pattern with the banding pattern
Are there any previously mapped genes in this region that provide some insight into your gene?